Peptide Library Production Method

ABSTRACT

The purpose of the present invention is to provide a method of producing a peptide library including a step of bringing a peptide library including peptides having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating at least a part of amino acid residues contained in at least some of the peptides.

The content of the ASCII text file of the sequence listing named “20211102_034574_029US1_subseq_ST25” which is 260 kb in size was created on Nov. 2, 2021 and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a method of producing a peptide library and a method of producing a peptide-mRNA complex library.

BACKGROUND ART

Research and development of various peptide drugs have recently been carried out. Peptide drugs have, as the largest advantage thereof, high affinity and high specificity to target molecules and ability of inhibiting protein-protein interaction which cannot be achieved by low molecular weight compounds. Due to chemical and biological diversity, peptide drugs tend to have higher specificity in interaction with target molecules than that of low molecular weight compounds and as a result, they can have greater physiological activity.

On the other hand, similar to most of other biopharmaceuticals, peptide drugs cannot penetrate a cellular membrane and therefore cannot reach the inside of cells and in addition, compared with large proteins such as antibodies, they are degraded in a short time due to their inferiority in protease resistance. They therefore have a problem with producing a sufficient effect. In recent years, improvement for such problems of peptide drugs has been investigated by subjecting peptides to various modifications.

The present inventors have developed a RAndom Peptide Integrated Discovery (RAPID) system as a protein translation system. This RAPID system is an experiment system that enables construction of a highly-diverse special cyclic peptide library and identification of active peptides from the library by using, in combination, an artificial translation synthesis system specialized in the synthesis of special cyclic peptides and a screening system making use of the characteristics of the translation system or molecular evolution engineering technique.

Since in the RAPID system, an arbitrary amino acid can be linked with an arbitrary tRNA by using an artificial aminoacylated RNA catalyst “flexizyme” (for example, Non-Patent Document 1), a desired amino acid can be bonded to tRNA having a desired anticodon. By making a desired amino acid correspond to an arbitrary codon different from that in a natural genetic code and performing codon reassignment for reprograming a genetic code table, it is possible to create a peptide library having an arbitrary amino acid containing a non-proteinogenic amino acid introduced at an arbitrary position of the peptide or a peptide library having a macrocyclic structure.

The highly-diverse special cyclic peptide library to be constructed includes peptides having enhanced protease resistance, cell permeability, or affinity or specificity to target molecules.

Further, by in vitro selection, peptides which will be active species that bind to an arbitrary target protein can be identified from the resulting library rapidly with high reliability.

CITATION LIST Non-Patent Document

-   Non-Patent Document 1: H. Murakami, H. Saito, and H. Suga, (2003),     Chemistry & Biology, Vol. 10, 655-662.

SUMMARY Technical Problem

Peptides that bind to an arbitrary target protein can be identified rapidly using a peptide library created with a RAPID system, but the fact is that peptides thus identified have still failed to have sufficient cell membrane permeability.

Also for the purpose of creating peptides having activity to a target in cells, there is therefore an eager demand for a method of producing a peptide library including peptides excellent in cell membrane permeability.

An object of the present invention is to provide a novel method of producing a peptide library. In particular, an object is to provide a method of producing a peptide library capable of producing a peptide library including peptides which are expected to have cell membrane permeability and have a highly hydrophobic local structure.

Solution to Problem

The present inventors have proceeded with investigation to overcome the above-described problem. As a result, it has been found that the above-described problem can be overcome by modifying, in a peptide library, a predetermined amino acid contained in peptides constituting the peptide library with a prenylation enzyme, leading to the completion of the present invention.

The present invention is as follows.

[1] A method of producing a peptide library including prenylated peptide(s), including a step of bringing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.

[2] The method of producing a peptide library as described above in [1], wherein the prenylation enzyme is at least one or more selected from the group consisting of Enzyme 1 to Enzyme 21 and homologs thereof.

[3] The method of producing a peptide library as described above in [1], wherein the prenylation enzyme has any amino acid sequence of the following (1) to (3):

(1) an amino acid sequences represented by any of SEQ ID NOS: 1 to 56,

(2) an amino acid sequence with one or more amino acid deletions, substitutions, or additions in an amino acid sequences represented by any of SEQ ID NOS: 1 to 56 having, and

(3) an amino acid sequence having 80% or more homology with an amino acid sequences represented by any of SEQ ID NOS: 1 to 56.

[4] The method of producing a peptide library as described above in any of [1] to [3], wherein the peptide library includes a cyclic peptide.

[5] The method of producing a peptide library as described above in [4], wherein the cyclic peptide has a cyclic structure formed by bonding of two amino acid residues through a disulfide bond, peptide bond, alkyl bond, alkenyl bond, ester bond, thioester bond, ether bond, thioether bond, phosphonate ether bond, azo bond, C—S—C bond, C—N—C bond, C═N—C bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, or triazole bond.

[6] The method of producing a peptide library as described above in any of [1] to [5], including, prior to the prenylation step, a step of translating an mRNA library in a cell-free translation system and preparing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof.

[7] A method of producing a peptide-genotype complex library, including: a step of preparing a peptide-genotype library including a complex between a genotype and a peptide, and a step of bringing the peptide-genotype library into contact with a prenylation enzyme to prenylate at least one Trp or derivative thereof.

[8] The method of producing a peptide-genotype complex library as described above in [7], wherein:

the peptide-genotype library preparation step includes a step of preparing a peptide-mRNA library by mRNA display method; and

the peptide-mRNA library preparation step includes:

a step of bonding a puromycin to a 3′-end of each mRNA of an mRNA library to produce a puromycin-bound mRNA library, and

a step of translating the puromycin-bound mRNA library in a cell-free translation system and preparing a peptide-mRNA library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof.

[9] A screening method for identifying a peptide binding to a target material, including a step of bringing the peptide library prepared by the production method described above in any of [1] to [6] and/or the peptide-genotype complex library prepared by the production method described above in [7] or [8] into a target material, and a step of selecting a peptide binding to the target material.

[10] A method of producing a prenylated peptide, including a step of bringing a peptide having an amino acid sequence containing at least one Trp or derivative thereof into a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.

[11] A method of producing a prenylated peptide as described above in [10], including a step of, prior to the prenylation step, synthesizing a peptide having an amino acid sequence containing at least one Trp or derivative thereof by chemical synthesis.

Advantage of the Present Invention

According to the present invention, a novel method of producing a peptide library can be provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a histogram showing, in the mass spectrum of a reaction product obtained by changing the amino acid residue present upstream of W in peptide YG326, followed by prenylation, the results of quantitatively determining the respective peak intensities of an unmodified peptide and a modified peptide and calculating an apparent modification efficiency on the mass spectrum.

FIG. 2 is a histogram showing, in the mass spectrum of a reaction product obtained by changing the amino acid residue present downstream of W in peptide YG326, followed by prenylation, the results of quantitatively determining the respective peak intensities of an unmodified peptide and a modified peptide and calculating an apparent modification efficiency on the mass spectrum.

FIG. 3 is a histogram showing the results of subjecting respective peptides containing the 10 kinds of motifs each composed of 3 amino acid residues to a prenylation reaction and calculating an apparent modification efficiency on a mass spectrum.

FIG. 4 is a histogram showing the results of subjecting mutants obtained by converting the X portion of VWX to the 6 kinds of amino acids (G, W, S, F, N and K) to a prenylation reaction and calculating an apparent modification efficiency on the mass spectrum.

FIG. 5 is a diagram showing that a desired DNA library is obtained as a product by PCR.

FIG. 6 is a diagram of a gel of electrophoresis showing the success in preparation of an mRNA library from a PCR product.

FIG. 7 is a diagram showing SDS-PAGE from which it has been confirmed that KgpF having a His tag can be immobilized onto NTA beads.

FIG. 8 shows a selection scheme of a prenylated cyclic peptide.

FIG. 9 is a graph of recovery rates of cDNAs in selection against MetAP1 used as a target.

FIG. 10 is a diagram showing sequences obtained by selection against MetAP1 used as a target. From top to bottom, the sequence identifiers in: the left column are SEQ ID NOs: 387 to 425; the middle column are SEQ ID NOs: 426 to 462; and the right column are SEQ ID NOs: 463 to 486.

FIG. 11 is a diagram showing the results of pulldown assay of prenylated cyclic peptides identified as a result of screening with MetAP1 as a target. From top to bottom, the sequence identifiers are SEQ ID NOs: 354 to 359, 354, and 355. At the bottom, middle of FIG. 11, the top sequence is SEQ ID NO: 354 and the bottom sequence is SEQ ID NO: 355.

FIG. 12 is a diagram showing a MALDI-TOF-MS spectrum before and after treatment with KgpF, of cyclic peptides chemically synthesized by Fmoc solid-phase synthesis. From top to bottom, the sequence identifiers are SEQ ID NOs: 360 to 364.

FIG. 13 is a diagram showing the results of MetAP1 enzymatic activity inhibition test by Met2, Met3a-L and Met3-1. From left to right, the sequence identifiers are SEQ ID NOs: 361, 362, and 360.

FIG. 14 is a diagram showing the results of the progress of a prenylation reaction of the cyclic peptide Kgp1-1 (SEQ ID NO: 365) by KgpF with or without Mg2+ in the presence of EDTA.

FIG. 15 is a diagram showing the results of the progress of a prenylation reaction of the cyclic peptide Met3-1 (SEQ ID NO: 360) by KgpF with or without Mg2+ in the presence of EDTA.

FIG. 16 is a diagram showing the results of the progress of a prenylation reaction of the cyclic peptide Met14 (SEQ ID NO: 381) by OltF with or without Mg2+ in the presence of EDTA.

FIG. 17 is a diagram showing MALDI-TOF-MS spectrum of a translated mixture containing an artificial cyclic peptide containing an amino acid in D-form before and after treatment with KgpF. From top to bottom, the sequence identifiers are SEQ ID NOs: 366 to 369.

FIG. 18 is a diagram showing the MALDI-TOF-MS spectrum of a cyclic peptide containing a non-proteinogenic Trp derivative after prenylation of the Trp derivative with OltF or KgpF.

FIG. 19 is a diagram showing the MALDI-TOF-MS spectrum of a cyclic peptide containing a Trp at two positions, that is, at N-terminal position and in the sequence, before and after treatment with OltF.

FIG. 20 is a diagram showing the MALDI-TOF-MS spectrum of a cyclic peptide containing a Trp at N terminal position, before and after treatment with OltF. From left to right, the sequence identifiers in: the top row are SEQ ID NOs: 372 and 373; the middle row are SEQ ID NOs: 374 and 375; and the bottom row are SEQ ID NOs: 376 and 377.

DESCRIPTION OF EMBODIMENTS

(Method of Producing Peptide Library)

The method of producing a peptide library according to the present invention includes a step of bringing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.

The peptide library in the present invention includes at least two peptides. At least one of the peptides included in the peptide library of the present invention has an amino acid sequence containing at least one Trp or derivative thereof.

In the present invention, a peptide library including a highly hydrophobic peptide can be produced by prenylating some of Trps or derivatives thereof with a prenylation enzyme. Since the prenylation enzyme has high substrate tolerance, hydrophobicity of the peptide included in the peptide library can efficiently increase. For the reasons described above, the production method of the present invention makes it possible to efficiently introduce a hydrophobic group into a peptide and thereby produce a peptide library including peptide(s) expected to have cell membrane permeability.

(Step of Preparing Peptide Library)

As the peptide library used in the production method of the present invention, a peptide library may be obtained by production or may be obtained by purchase. When the peptide library is obtained by production, preferred examples of the production method include a method of producing a peptide library by solid-phase or liquid-phase chemical synthesis and a method of translating an mRNA library in a cell-free translation system and thereby preparing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof.

One of preferred aspects of the production method of the present invention includes a step of translating an mRNA library in a cell-free translation system and thereby preparing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof and a step of bringing the peptide library into contact with a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.

The mRNA library contains an mRNA having a plurality of N1N2N3s as a codon.

The term “N1N2N3” as used herein means a codon specifying an arbitrary amino acid and for example, N1, N2, and N3 are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U). One mRNA contains a plurality of N1N2N3s and the N1, N2 and N3 are each independently selected. For example, when mRNA has —N1N2N3-N1N2N3-, two N1s, N2s, and N3s may be the same or different from each other.

In the present invention, an arbitrary amino acid is reassigned to N1N2N3. In the reassignment, a codon-amino acid relation different from that in a natural genetic code table can be reassigned or alternatively, the same relation can be reassigned.

The term “natural genetic code table” means a table showing amino acids represented by genetic codes composed of an mRNA triplet in a living body. In the natural genetic code, N1N2N3 represents the following amino acids.

TABLE 1 U C A G U Phe Ser Tyr Cys U Phe Ser Tyr Cys C Leu Ser STOP STOP A Leu Ser STOP Trp G C Leu Pro His Arg U Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G A Ile Thr Asn Ser U Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G G Val Ala Asp Gly U Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G

The mRNA library may include an mRNA containing, as the plurality of N1N2N3s, for example, any of a plurality of N1N2Ks, a plurality of N1N2Ss, a plurality of N1N2Ms, a plurality of N1N2Ws, a plurality of N1N2As, a plurality of N1N2Us, a plurality of N1N2Cs, and a plurality of N1N2Gs. In the present specification, N1 and N2 each have the same meaning as described above, Ks each independently represent either uracil (U) or guanine (G), Ss each independently represent either cytosine (C) or guanine (G), Ms each independently represent either adenine (A) or cytosine (C), and Ws each independently represent either adenine (A) or uracil (U).

For convenience, the present invention will next be described using, as an example, an mRNA library including mRNA having a plurality of N1N2Ks, that is, an mRNA library including mRNA having, as a codon, N1N2Ks as N1N2N3. Even if the other mRNA library is used, it may be possible insofar as a translated peptide library contains a peptide to be prenylated. In the natural genetic code table, N1N2K represents 20 kinds of amino acids with G or U in the right column of the table.

In the present specification, for example, Leu may be assigned to UUG as shown in the natural genetic code table or an amino acid other than Leu may be assigned by codon reassignment. Any amino acid can be assigned to a “N1N2K” codon. The term “an amino acid is assigned to a codon” means that a genetic code table is rewritten so that a certain codon encodes the amino acid. The term “an amino acid is assigned to a codon” and the term “a codon is reassigned” have the same meaning in the present specification.

Assignment of an amino acid to each codon different from that of the natural genetic code table is achieved by codon reassignment making use of, for example, an artificial aminoacylated RNA catalyst flexizyme. Using the flexizyme enables bonding of a desired amino acid to a tRNA having an arbitrary anticodon so that an arbitrary amino acid can be assigned to an arbitrary codon. The flexizyme will be described later. In the present invention, “bonding of an amino acid to tRNA” is also expressed by “charging an amino acid with tRNA”, “aminoacylating tRNA”, or “acylating tRNA with an amino acid”.

In the present invention, a non-proteinogenic amino acid may be assigned to “N1N2K”. For example, using, as a non-proteinogenic amino acid, an amino acid having a cyclic structure or an N-alkylamino acid makes it possible to obtain a peptide library having enhanced resistance to proteolysis, cell membrane permeability, and conformational rigidity. Such a peptide library is useful for the screening of peptides targeting an intracellular disease-related molecule or a molecule having a protease activity. When mRNA contains two or more “N1N2Ks”, all of them may be assigned to a non-proteinogenic amino acid or some of them may be assigned to a non-proteinogenic amino acid.

The term “amino acid” as used herein is used in its broadest meaning and it embraces not only natural amino acids but also artificial amino acid mutants and derivatives. The amino acids may be represented by a commonly used single-letter or three-letter code. Examples of the amino acid or derivatives thereof used herein include natural proteinogenic L-amino acids, unnatural amino acids, and chemically synthesized compounds having properties known in the art as characteristics of an amino acid. Examples of the unnatural amino acids include, but not limited to, α,α-disubstituted amino acids (such as α-methylalanine), N-alkyl-α-amino acids, D-amino acids, β-amino acids, and α-hydroxy acids, each having a main chain structure different from that of natural amino acids; amino acids (such as norleucine and homohistidine) having a side-chain structure different from that of natural amino acids; amino acids (such as “homo” amino acids, homophenylalanine, and homohistidine) having extra methylene in the side chain thereof; and amino acids (such as cysteic acid) obtained by substituting the functional group of a carboxylic acid in the side chain of the amino acids by a sulfonic acid group.

The term “amino acid” as used herein embraces proteinogenic amino acids and non-proteinogenic amino acids. The term “proteinogenic amino acid” as used herein means an amino acid (Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr or Val) constituting a protein.

The term “non-proteinogenic amino acid” as used herein means a natural or unnatural amino acid other than the proteinogenic amino acid.

The derivative of Trp is represented, for example, by the following formula (I), formula (II), or formula (III). It is to be noted that the chemical structural formula shown herein embraces tautomers, geometrical isomers, optical isomers, and the like. In particular, asymmetric carbon in the formula (I), formula (II), or formula (III) may have either an R configuration or an S configuration, or a mixture of them.

The ring A in the formula (I) is an aromatic 6-membered ring which may contain a hetero atom; R1 is an arbitrary substituent on the ring A; R² and R³ each independently represent a hydrogen atom or an arbitrary substituent; m stands for any integer of from 0 to 4, and l stands for an integer of 1 or 2.

Examples of the ring A include benzene ring, pyridine ring, pyridazine ring, pyrimidine ring, and pyrazine ring.

Although the substituents R¹, R², and R³ are not particularly limited, examples include a hydroxy group; halogen groups such as fluorine group, chloro group, bromo group, and iodo group; alkyl groups having from 1 to 10 carbon atoms such as methyl group, ethyl group, and propyl group; aryl groups such as phenyl group and naphthyl group; aralkyl groups such as benzyl group; and aralkyloxy groups such as benzyloxy group.

In the formula (II), the ring A, R¹, R², m, and l have the same meanings as the ring A, R¹, R², m, and l in the formula (I). The ring A in the formula (II) is preferably a benzene ring.

In the formula (III), the ring A, R¹, R², m, and l have the same meanings as the ring A, R¹, R², m, and l in the formula (I). The ring A in the formula (III) is preferably a benzene ring.

Examples of the Trp derivative represented by the formula (I) include the following derivatives.

In the above derivatives, R¹, R², R³ and m have the same meanings as R¹, R², R³, and m in the formula (I).

Specific examples of the derivatives of Trp include D-tryptophan, 5-methyltryptophan, 5-hydroxytryptophan, 7-azatryptophan, and 5-benzyloxytryptophan.

It is presumed that since the Trp residue is prenylated to form a structure having an alkylated main-chain nitrogen as shown in the following formula (T1), a hydrogen bond donor decreases and the peptide containing the Trp residue tends to have improved hydrophobicity and membrane permeability (Refer to Beilstein J Org Chem. 2017 Feb. 22; 13:338-346.).

Here, the prenyl group is a structural unit composed of C₅ isoprene unit(s) and it is the following structure portion when the formula (T1) is used as an example.

(In the above structure, n stands for any integer of from 0 to 11).

Although the steric configuration in the formula (T1) is not particularly limited, examples include steric configurations represented by the following formula (T1-1) and (T1-2). The steric configuration in the formula (T1) preferably has the steric configuration represented by the following formula (T1-1).

When the derivative residue of Trp is prenylated, the resulting product can be represented by the following formulas (I-1), (II-1), and (III-1).

(In the formulas (I-1), (II-1), and (III-1), the ring A, R¹, R², R³, m, and l have the same meanings as the ring A, R¹, R², R³, m, and l in the formula (I), respectively).

By the method of producing a peptide library according to the present invention, a peptide library including several kinds of peptides including an amino acid encoded by “N1N2N3” can be produced. The number of amino acids encoded by “N1N2N3” included in each peptide is not particularly limited, it can be set at, for example, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30. The position of an amino acid encoded by “N1N2N3” in each peptide is not particularly limited. Amino acids encoded by “N1N2N3” in each peptide may be adjacent to each other or may be separated from each other.

In the method of producing a peptide library according to the present invention, an mRNA library including mRNA(s) which encode each peptide of the peptide library, respectively, and each of which contains at least one N1N2N3 is prepared.

The sequence of mRNAs which encode the peptides of the peptide library, respectively, can be determined depending on the amino acid sequence of the peptides constituting the peptide library and such an mRNA library can be prepared by synthesizing a DNA library encoding it and transcribing the resulting library.

For example, as shown below in Table 2, an mRNA library including mRNAs having, as N1N2N3, NNG, NNK, NNS, NNM, NNW, NNA, NNU or NNC can be designed while making use of the natural genetic code table.

TABLE 2 AUG (NNG)n UGU (SEQ ID NO: 491) AUG (NNK)n UGU (SEQ ID NO: 492) AUG (NNS)n UGU (SEQ ID NO: 493) AUG (NNM)n UGU (SEQ ID NO: 494) AUG (NNW)n UGU (SEQ ID NO: 495) AUG (NNA)n UGU (SEQ ID NO: 496) AUG (NNU)n UGU (SEQ ID NO: 497) AUG (NNC)n UGU (SEQ ID NO: 498)

In Table 2, the Ns are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U).

In the above table, Ks each independently represent either uracil (U) or guanine (G);

Ss each independently represent either cytosine (C) or guanine (G);

Ms each independently represent either adenine (A) or cytosine (C); and

Ws each independently represent either adenine (A) or uracil (U).

In the above table, n is an arbitrary integer of 1 or more, preferably any integer of from 1 to 100, more preferably any integer of from 3 to 50, still more preferably any integer of from 3 to 20, still more preferably any integer of from 6 to 15.

When the natural genetic code table is used, a triplet encoding Trp is only one kind, that is, UGG, among 64 kinds of N1N2N3s. In theory, this suggests that the probability of one N1N2N3 triplet encoding Trp is 1 in 64. On the other hand, for example, the probability of one NNK triplet encoding Trp is 1 in 32. An mRNA library including, for example, mRNA(s) having 12 triplet repeats and having NNK as N1N2N3 can be designed while making use of the natural genetic code table. Such an mRNA library theoretically has diversity as high as 1.15×10¹⁸ and includes 22% mRNAs not only containing no stop codon but also containing at least one UGG.

For example, the probability of one NNG triplet encoding Trp is 1 in 16. Such an mRNA library including NNG triplets and having n of 12 theoretically has diversity as high as 2.8×10¹⁴ and includes 26% mRNAs not only containing no stop codon but also containing at least one UGG.

The present inventors have found that in the prenylation of a Trp residue with a prenylation enzyme, an amino acid residue upstream or downstream of the Trp residue has an influence on a modification efficiency. By using an mRNA library including mRNA(s) containing a codon encoding an amino acid residue which is adjacent to a Trp or derivative thereof to be prenylated and promotes the prenylation of the Trp or derivative thereof, the prenylation efficiency of the Trp or derivative thereof can be improved.

For example, as shown below in Table 3-1, using an mRNA containing UGG and a codon (XXX, YYY) adjacent to UGG and encoding an amino acid residue that promotes prenylation enables efficient introduction of a prenyl group in a peptide library.

TABLE 3-1 AUG (N1N2N3)n XXX UGG (N1N2N3)m UGU (SEQ ID NO:  499) AUG (N1N2N3)n UGG YYY (N1N2N3)m UGU (SEQ ID NO:  500) AUG (N1N2N3)n XXX UGG YYY (N1N2N3)m UGU (SEQ ID  NO: 501)

In Table 3-1, XXX is a degenerate codon sequence that specifies a group of upstream amino acids which become more susceptible to a prenylation enzyme used.

YYY is a degenerate codon sequence that specifies a group of downstream amino acids which become more susceptible to a prenylation enzyme used.

In the above table, n and m each independently represent an any integer of 0 or more and the sum of n and m is preferably any integer of from 1 to 100, more preferably any integer of from 3 to 50, still more preferably any integer of from 3 to 20, still more preferably any integer of from 4 to 13.

N1N2N3 has the same meaning as defined above and is, for example, a codon selected from the group consisting of NNK, NNS, NNW, NNM, NNA, NNC, NNG, and NNU. Two or more N1N2N3s are each a codon independently selected.

A modification efficiency with a prenylation enzyme changes depending on the kind of a prenylation enzyme used and it can be enhanced by adjusting an upstream and/or downstream amino acid residue adjacent to Trp (XXX, YYY as a corresponding codon). The amino acid sequence(s) for efficiency enhancement composed of Trp and the upstream and/or downstream amino acid residue(s) adjacent to Trp, XXX and YYY can be determined as needed by comprehensively preparing peptides in a cell-free translation system and carrying out an efficiency confirmation test by performing in vitro prenylation with a predetermined prenylation enzyme.

When KgpF is used as a prenylation enzyme, prenylated Trp can be introduced into a peptide efficiently by being contained the mRNAs shown in Table 3-2, among mRNAs in Table 3-1, in a library.

TABLE 3-2 AUG (NNK)n XXX UGG (NNK)m UGU (SEQ ID NO: 502)

In Table 3-2, XXX, NNK, n, and m have the same meanings as described above.

Using an mRNA library including such a mRNA as the mRNA shown in Table 3-2, having an n and m of arbitrary integer and containing UGG while making use of the natural genetic code table makes it possible to fix the appearance position of UGG in the resulting peptide library and control the position of the Trp residue to be prenylated in the peptide. Further, when, for example, KgpF is used as a prenylation enzyme, the prenylation efficiency of the Trp residue can be made higher by designating the triplet XXX upstream of UGG to RYU or CMG and/or designating the triplet downstream of UGG to NNK. Such an mRNA library theoretically has diversity as high as 6.8×10¹⁶ and includes 73% mRNAs not only containing no stop codon but also containing at least one UGG.

In Table 3-2, when XXX represents RYU or CMG, Rs each independently represent G or A, Ys each independently represent U or C, Ms each independently represent A or C, and Ks each independently represent G or U.

In the method of producing a peptide library according to the present invention, the mRNAs of the mRNA library are each translated in a cell-free translation system having, for example, 20 kinds of tRNAs having an anticodon for any of 20 N1N2N3 codons and charged with an amino acid corresponding to the codon.

For codon reassignment, a translation system obtained by arbitrarily removing a component constituting a translation system depending on a purpose and then reconstituting with only necessary components can be used. For example, when a translation system from which a specific amino acid has been removed is reconstituted, a codon corresponding to the amino acid becomes an empty codon, that is, a codon not encoding any amino acid. An arbitrary amino acid is then linked to a tRNA having an anticodon complementary to the empty codon by making use of flexizyme or the like. After addition of the resulting tRNA, translation is performed. The arbitrary amino acid is then encoded by the codon and a peptide having the arbitrary amino acid introduced therein instead of the removed amino acid is translated.

The tRNA to be used in the present invention may be a wild-type Escherichia-coli derived tRNA or an artificial tRNA prepared by in vitro transcription.

In the present invention, 20 kinds of tRNAs corresponding to 20 kinds of N1N2N3 to be used in the translation system may have the same sequence except for an anticodon loop portion. Using such a constitution enables the tRNAs to have uniform reactivity without enhancing or reducing the reactivity of a specific tRNA, leading to expression of a predetermined peptide with good reproducibility.

The term “cell-free translation system” as used herein means a translation system not containing cells. As the cell-free translation system, for example, an Escherichia coli extract, a wheat germ extract, a rabbit reticulocyte extract, or an insect cell extract can be used. Also usable is a reconstituted cell-free translation system constructed by the reconstitution of ribosome protein, aminoacyl-tRNA synthetase (aaRS), ribosomal RNA, amino acid, rRNA, GTP, ATP, translation initiation factor (IF), elongation factor (EF), release factor (RF), and ribosome regeneration factor (RRF), and another factor necessary for translation, each of which has been purified.

The system may contain an RNA polymerase for simultaneously performing transcription from DNA. As a commercially available cell-free translation system, usable are Escherichia-coli derived systems such as RTS-100 (registered trademark) of Roche Diagnostics, reconstituted translation systems such as PURESYSTEM (registered trademark) of PGI, PUREfrex of GeneFrontier, PURExpress In Vitro Protein Synthesis Kit of New England Biolabs, and systems using a wheat germ extract such as those of ZOEGENE and CellFree Sciences.

As a system using a ribosome of Escherichia coli, for example, techniques described in the following documents are known: H. F. Kung et al., 1977. The Journal of Biological Chemistry Vol. 252, No. 19, 6889-6894; M. C. Gonza et al., 1985, Proceeding of National Academy of Sciences of the United States of America Vol. 82, 1648-1652; M. Y. Pavlov and M. Ehrenberg, 1996, Archives of Biochemistry and Biophysics Vol. 328, No. 1, 9-16; Y. Shimizu et al., 2001, Nature Biotechnology Vol. 19, No. 8, 751-755; H. Ohashi et al., 2007, Biochemical and Biophysical Research Communications Vol. 352, No. 1, 270-276.

By using the cell-free translation system, a highly-pure expression product can be obtained without purification.

The cell-free translation system of the present invention may be used for not only for translation but also for transcription after addition of factor(s) necessary for transcription.

In the present invention, it is preferred not to add a natural aminoacyl-tRNA synthetase corresponding to an N1N2K codon to the cell-free translation system. In this case, the tRNA corresponding to the N1N2K codon can be charged with a corresponding amino acid, for example, by an artificial aminoacyl-tRNA synthetase such as flexizyme.

The peptide library in the present invention may include a cyclic peptide obtained by cyclization of a peptide. The term “cyclic peptide” as used herein means a peptide having a cyclic structure composed of 4 or more amino acids. The term “cyclic structure” means, in a linear peptide, a closed ring structure formed in the molecule by bonding, directly or via a linker or the like, of two amino acids separated from each other by two or more amino acid residues. The term “separated from each other by two or more amino acid residues” means that the two amino acids have at least two amino acid residues therebetween.

The cyclic structure is formed by bonding of two amino acids through a disulfide bond, peptide bond, alkyl bond, alkenyl bond, ester bond, thioester bond, ether bond, thioether bond, phosphonate ether bond, azo bond, C—S—C bond, C—N—C bond, C═N—C bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, triazole bond, or the like. The kind of the bond is however not limited to them.

Cyclization of a peptide is sometimes useful for stabilizing the structure of the peptide and enhancing its affinity for a target.

The number of amino acids constituting a cyclic structure, in other words, amino acids forming a ring in the cyclic peptides is not particularly limited insofar they are four amino acids or more. They may be, for example, 4 amino acids or more, 5 amino acids or more, 8 amino acids or more, 15 amino acids or less, 20 amino acids or less, 25 amino acids or less, 30 amino acids or less, or the like.

The cyclization is not limited to that formed by bonding of the N-terminal and C-terminal amino acids of a peptide, but it may be formed by bonding of a terminal amino acid and a non-terminal amino acid or bonding of non-terminal amino acids. When one of the amino acids bonded for ring formation is a terminal amino acid and the other one is a non-terminal amino acid, the resulting cyclic peptide has a cyclic structure having a linear peptide attached thereto like a tail. Such a structure is sometimes called “lasso type” herein.

The term “amino acids adjacent to each other” as used herein means amino acids adjacent to each other even after a ring is formed. Amino acids adjacent to each other are, in a peptide, amino acids which are not separated from each other by one or more amino acid residues and in which amino acid residues have bonded to each other directly.

For cyclization, for example, a chloroacetylated amino acid may be used as the N terminal and a Cys may be placed downstream (on the C-terminal side) of the N terminal. In this case, through thioether bonding between the N-terminal amino acid and the Cys placed downstream (on the C terminal side) of the N terminal, spontaneous cyclization of the peptide occurs after expression. A thioether bond formed between a chloroacetylated amino acid and a Cys is not susceptible to degradation even under reducing conditions in a living body so that a physiologically active effect can be continued while prolonging the half-life in blood of the peptide.

Examples of the chloroacetylated amino acid include N-chloroacetyl-L-alanine, N-chloroacetyl-L-phenylalanine, N-chloroacetyl-L-tyrosine, N-chloroacetyl-L-tryptophan, N-3-chloromethylbenzoyl-L-phenylalanine, N-3-chloromethylbenzoyl-L-tyrosine, N-3-chloromethylbenzoyl-L-tryptophane, N-3-(2-chloroacetamido)benzoyl-L-phenylalanine, N-3-(2-chloroacetamido)benzoyl-L-tyrosine, and N-3-(2-chloroacetamido)benzoyl-L-tryptophane, and D-amino acid derivatives corresponding thereto.

Using, for example, Nγ-(2-chloroacetyl)-α,γ-diaminobutyric acid or Nγ-(2-chloroacetyl)-α,γ-diaminopropanoic acid as the chloroacetylated amino acid enables introduction of itself into any site of the peptide chain so that a thioether bond is formed between the amino acid at any position and a cysteine in the same peptide to form a cyclic structure.

The cyclization method may be carried out according to the method described, for example, in Kawakami, T. et al., Nature Chemical Biology 5, 888-890 (2009); Yamagishi, Y. et al., Chem Bio Chem 10, 1469-1472 (2009); Sako, Y. et al., Journal of American Chemical Society 130, 7932-7934 (2008); Goto, Y. et al., ACS Chemical Biology 3, 120-129 (2008); and Kawakami T. et al, Chemistry & Biology 15, 32-42 (2008).

The chloroacetylated amino acid and Cys may be bound to the peptide of the present invention directly or may be bonded via a linker or the like.

When the C-terminal amino acid of the cyclic peptide is not used for cyclization, this C terminal is not limited to a carboxyl group or a carboxylate group but may also be in the form of an amide or ester. The cyclic peptide of the present invention embraces salts of the cyclic peptide. Examples of the salts of the cyclic peptide include salts with a physiological acceptable base or acid such as addition salts of an inorganic acid (such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, or phosphoric acid), addition salts of an organic acid (such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, or acetic acid), inorganic bases (such as ammonium hydroxide, alkali or alkaline earth metal hydroxides, carbonates, or bicarbonates), and addition salts of an amino acid.

The cyclic peptide of the present invention may be those modified through phosphorylation, methylation, acetylation, adenylylation, ADP ribosylation, glylcosylation or the like insofar as it solves the problem of the present invention. It may be those fused with another peptide or protein.

(Prenylation Step)

The method of producing a peptide library according to the present invention includes a step of bringing a peptide library into contact with a prenylation enzyme and prenylating at least a part of Trps or derivatives thereof included in at least some of peptides. The term “prenylation enzyme” as used herein means an enzyme capable of bonding a prenyl group composed of a C₅ isoprene unit to at least a part of Trps or derivatives thereof contained in the peptide. The term “prenyl group” as used herein embraces a group having a plurality of isoprene units consecutively bonded to each other. Prenyl groups having 1, 2, 3, 4, 5, 6, 8, and 10 isoprene units may be called “dimethylallyl group”, “geranyl group”, “farnesyl group”, geranylgeranyl group”, “geranylfarnesyl group” “hexaprenyl group”, “octaprenyl group”, and “decaprenyl group”, respectively.

In the prenylation step in the method of producing a peptide library according to the present invention, a peptide library is preferably brought into contact with a prenylation enzyme in the presence of a donor for a prenyl group. The donor for providing a prenyl group is not particularly limited and examples include dimethylallyl diphosphate (DMAPP), isoprenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and phytyl diphosphate (PDP). These prenyl group donors may be used either singly or in combination of two or more.

The prenylation step in the method of producing a peptide library in the present invention may be performed in the presence or absence of a cofactor. The term “cofactor” as used herein means a material capable of enhancing the catalytic activity of an enzyme. Although the cofactor is not particularly limited, examples include magnesium ion, copper ion, iron ion, manganese ion, molybdenum ion, nickel ion, selenium ion, zinc ion and the like. They may be used either singly or in combination of two or more. Of these, magnesium ion is preferred.

Whether the cofactor is used or not in the method of producing a peptide library according to the present invention may be selected as needed, depending on the prenylation enzyme used. When a peptide library to be subject to prenylation is prepared in a cell-free translation system, prenylation can be performed without adding a cofactor because cofactor(s) is sometimes contained in this cell-free translation system.

Specific examples of the prenylation enzyme usable in the present invention include KgpF, OltF, TruF1, TruF2, PatF, LynF, AcyF, OscF and PagF, and homologs thereof capable of prenylating a Trp residue or Trp derivative, each listed below in Tables 4-1 to 4-2. These prenylation enzymes can modify a very wide range of substrates regardless of the kind of amino acid residues adjacent to a Trp or derivative thereof to be prenylated, as verified in Examples described later. The term “amino acid derivative” as used herein is used in its broadest sense. It embraces any amino acids other than natural amino acids and also includes the above-described artificial amino acid mutants and derivatives. The prenylation enzyme in the present invention can prenylate even an amino acid residue contained in a cyclic peptide so that also from this standpoint, it can modify a very wide range of substrates.

The prenylation enzyme can also be used while being supported on a solid-phase support such as magnetic beads. The prenylation enzyme in the present invention therefore embraces an aspect of an enzyme supported on a solid-phase support. The prenylation enzyme in the present invention may have an addition sequence of His tag, glutathione-S-transferase (GST), maltose-bound protein (MBP), or the like.

The prenylation enzyme and homologs thereof capable of prenylating a Trp or derivative thereof are not particularly limited insofar as they can prenylate a Trp or derivative thereof. As the prenylation enzyme, preferred is, as a result of selecting a known prenylation enzyme (enzyme obtained as a result of BLAST search analysis), forming a sequence alignment by using, for example, a Clusta IW, MEGA7 software, and performing phylogenetic analysis, an enzyme having an amino acid sequence found to have homology with the amino acid sequence of this known prenylation enzyme. The enzyme having an amino acid sequence found to have homology as a result of analysis includes a protein annotated as hypothetical protein.

As the prenylation enzyme, for example, Enzyme 1 to Enzyme 21 listed in Table 4-1 and Table 4-2 can be used. These enzymes may be used in combination of two or more.

Homologues of the prenylation enzymes from Enzyme 1 to Enzyme 21 are not particularly limited insofar as they are enzymes serving to prenylate a Trp or derivative thereof and they may be enzymes having an amino acid sequence having 20% or more homology with the alignment of Enzyme 1 to Enzyme 21.

The prenylation enzyme in the present specification is not particularly limited insofar as it has at least a domain engaged in a prenylation reaction. The homologs of Enzyme 1 to Enzyme 21 may be an aspect of an enzyme having a domain engaged in the prenylation reaction and a domain engaged in the other function (such an enzyme may also be called “bi-functional enzyme”) or may be an aspect of two or more enzymes each having a domain engaged in prenylation.

Of the enzymes listed in Table 4-1 and Table 4-2, enzymes of Group 1 and Group 2 are preferred, with enzymes of Group 1 being more preferred.

TABLE 4-1A SEQ Group Enzyme accession No. name or annotation ID NO: amino acid sequence 1 1 WP_061432709.1 KgpF or LynF/TruF/ 1 MINYANAQLHKSKNLMYMKAHENIFEIEALYPL PatF family peptide O- ELFERFMQSQTDCSIDCACKIDGDELYPARFSL prenyltransferase ALYNNQYAEKQIRETIDFFHQVEGRTEVKLNYQ [Microcystis aeruginosa] QLQHFLGADFDFSKVIRNLVGVDARRELADSRV KLYIWMNDYPEKMATAMAWCDDKKELSTLIVNQ EFLVGFDFYFDGRTAIELYISLSSEEFQQTQVW ERLAKVVCAPALRLVNDCQAIQIGVSRANDSKI MYYHTLNPNSFIDNLGNEMASRVHAYYRHQPVR SLVVCIPEQELTARSIQRLNMYYCMN 2 WP_052338757.1 OltF or LynF/TruF/ 2 MTYPSLLTASQKNLQFIGEHKNAFDLEYLYPLD PatF family peptide O- LFEDLVAQVEPCRIECSCKIENNKLDPARFNLA prenyltransferase INDSFARKAQAFLGFFHQVETRVGVIIDYKPLH [Planktothrix] FFMKGFDYSKVTQIMAGIDARAELTEARVKTWW AIDNYPEKLETAIALSQSDSKPLRMLLEINNSL VIGFHLYLRGKSEVRLYPSVSREDFKKVSVQAQ LAKILSTPALKLLEDSVAISLVLKENGDKILHY HVLPERVNSFVNNLHQDMITRAHAPYRNKSQSL GCTVSLSEKELLAGYLKTINLYYMNT 2 3 WP_069351383.1 LynF/TruF/PatF family 3 MITDKHTLVDTNKNLHYIGEHKRAFQVESVYPL peptide O- DIFERFVQEATDWGLECSCKIEKDKLYPIRFNL prenyltransferase FRNHPSFKQFDAALDFFQQVEARDDVKLDYKLM [Scytonema millei] HLFLGNYFDFNKIAQILVGVDLRTELSASRLKL WFVIQNYPEKLETAFALCDLKEELRALIVCCSL VVVGFDFYLDGRTNIELYPRILKKELQEVDVWK QLAKVVSPPTLQLLDSCWAFMLGFSKANPETIL YCPARDPDSFIANLSNDLADRVHAYYQKQPVRG TIVAFRERELLAGAIENLNLYYQMSLGVTKKSE KFDGCVAKTGEAGADIQK 4 CEJ46811.1 Anacyclamide synthesis 4 MTISENLFIGSQKNLHYINQHKHIFDVDHLYTL protein AcyF NIFDDFAQKVTHWGLECSCKIQKDQLYPARFNL [Chrysosporum FRNQPNWQDYQTAINFFQQVAAKTDVKLTYHLI ovalisporum] ENFVGNDFDFSKVHQILVGIDLRREFSASRLKF WFIISDYPQKLAKAISLCQSAEELQPFLVDNSV VIGFDFDFNGGSEIEVYPSISKEKFQQLAKVLS PQALQLLDSCWSLIIGFSQGNAEKILYYRTSDP NNFIANLRNDLANRVHAYYREQPVQGTIVGLRE NEIIAGRIENLNLYYQMSSPITTNKN 5 WP_028090374.1 LynF/TruF/PatF family 5 MTISENLFIGSQKNLHYINQHKHIFDVDHLYTL peptide O- NIFDDFAQKVTHWGLECSCKIQKDQLYPARFNL prenyltransferase FRNQPNWQDYQTAINFFQQVAAKTDVKLTYHLI [Dolichospermum circinale] ENFVGNDFDFSKVHQILVGIDLRREFSASRLKF WFIISDYPQKLAKAISLCQPAEELQPFLVDNSV VIGFDFDFYGGSQIEVYPSISKEKFQQLAKVLS PQALQLLDSCWSLIIGFSQANPEKILYYRTSDP NNFIANLRNDLANRVHAYYREQPVQGTIVGLRE NEIIAGRIENLNLYYQMSSPITTNKN 6 WP_099072935.1 LynF/TruF/PatF family 6 MVTYKPIQVDSEPYLEYIGKHKRAFDIYEDLYP peptide O- VKLFEDFVQVEGRQGGMFRCGCNVTKDELSPAR prenyltransferase FALLFPTQDDLQSLHNPQQQLEAAFDFFRKVEG [Nostoc linckia] RAEVKVNYHLLEKFFGKNPDFGGIYFIAVGVDA QTEISESRLKLFIHLHDAPDKIESAIALSGDSP TLRAFLVNNDLLVGFDFYLDGRSEIEVYPTIYQ EELKRADIQSRILPLLPPRALPLLKQCEVLQIG ISPANKSNILYFDFVRDPNTFIDNLGNEMAKKV HAYYRHQPVQHVLVGVSEKDLYASSIEKVKLYY FKK

TABLE 4-1B 2  7 WP_099072934.1 LynF/TruF/PatF  7 MVTYNPIKTNSERYLEYIGKHKLAFDIYEDLYPLKIFEDFVEVEAK family peptide O- KRGLFYILSNVDKDEIYPARFCLRFPSLEEAQLLYNPQQQLQTALN prenyltransferase FFRQVESRPEVKLNYHHIQQFFGSISDFQGIILMAVAIDARTVITE [Nostoc linckia] SRLKLYVWLKNAPEKVETAIALCGDSPTLRAFLVNDKLQVGFDLFF NGESEIEVYPIISQDELQQVHIRDRIIPLLPPRALPLLQQCAVFQV GFSEANESNILYFDYVHDPNSFVDNLGNEMTKKIHAYYRHQPIKSL TVGIPEHNFYGRAIEHVKLYYDMN  8 WP_061432522.1 LynF/TruF/PatF  8 MVLKSNKKLYYISANKHAFEIDNLYPLNLFEGFVERIEKIEKTENC family peptide O- VLESSCKIDHDKLYPVRFNIGFPNNSIKQLHAVMDFFRRVESRVDV prenyltransferase KLNLSLFQQFIGNDFKLDKMTDLMLGIDLRRDLSDSRLKIGLTIED [Microcystis YPEKQKAAVILNNNIDEVTSNLLISNRLHIGFDFYLNGRSEMELYP aeruginosa] HIMQQDFQKLDVQQRLSKVLSPPALQVVPACTRICVGISKANRDKI IYYYLENMGDFLNYFTVNDTARKVHAYYLKQPVVEMCVALPESELL AGTTIKNLNLYYLL  9 WP_035367793.1 LynF/TruF/PatF  9 MTTLLESGKKIYYIGIHKQIFEIKNFYPLDIFDSFVNQIETTSENC family peptide O- SLESSCKIELDKLYPARFGIGFTLKNLKQLNVVYEFFQKVESRIDV prenyltransferase QINYSLIQQFFGENFDFNKMTEFMVGIDARQELSETKLKIALTIKN [Dolichospermum YPEKIKTAIALNGGLDKNIYNLLVSNSLHIGFDLSLDGRSEIELYP circinale] YIRNQEFQIFDIQQRLATVLSPQALQFLPICSRICVGLSKANADKV VYFYLKNLNDFLNYFTVNDTARRVHAYYQQQPMREMCVAVQEKQLL GGTIEKMNLYYLI 10 WP_052345004.1 LynF/TruF/PatF 10 MTYPSLLTASQKNLQFIGEHKNAFDLEYLYPLDLFEDLVAQVEPCR family peptide O- IECSCKIEDDKLDPARFNLAINDSFARKAQAFLGFFHQVETRVGVI prenyltransferase IDYKPLHFFMKGFDYSKVTQIMAGIDARAELTEARVKTWWAIDNYP [Planktothrix EKLETAIALSQSDSKPLRMLLEINNSLVIGFHLYLRGKSEVRLYPS rubescens] VGREDFQKISVQAQLAKIISTPALKLLEDSGAISLVLKENGDKILH YHVLPERVNSFVDNLHQDMITRAHAPYRNKSQSLGCTVSLSEKELI AGSLKTINLYYMNQ 11 WP_027254539.1 LynF/TruF/PatF 11 MTHPSLLTVSQKNLQFIGEHKNAFDLEYLYPLDLFEDLVAQVEPCR family peptide O- IECSCKIENNKLDPARFNLAINDSFARKAQAFLGFFHQVETRVGVI prenyltransferase IDYKPLHFFMKGFDYSKVTQIMAGIDARAELTEARVKTWWAIDNYP [Planktothrix EKLETAIALSQSDSKPLRMLLEINNSLVIGFHLYLRGKSEVRLYPS agardhii] VSREDFKKVSVQAQLAKILSTPALKLLEDSVAISLVLKKNGDKILH YHVLPERVNSFVDNLHQDMITRAHAPYRNKSQSLGCTVSLSEKELI AGSLKTINLYYMNQ

TABLE 4-2A SEQ Group Enzyme accession No. name or annotation ID NO: amino acid sequence 2 12 WP_002767616.1 LynF/TruF/PatF 12 MATSLESSKKNYYISTHKQAFDVENFYPLEIWEKFV family peptide O- EKIEKTSEKCFLESSCKIDQGQFYAARFGIGFDLQN prenyltransferase LQQLNAVYNFCQEVESRVGVRVDYSLIKQFLGDDFD [Microcystis  FSKMTEFLIGVDARRELAESKLKIALTINDYPEKLK aeruginosa] TAIYLNGGLDETIEKLIVSNSLHLGFDLSLNGISEI ELYPYIGKQDFQRIDIQQRLATVLSPQALRPLAACR RICVGLSKGNTEKILYYYLEDIKDFLNYFTPNDTAR RVHAYYQKQPISEMCVAAPESQFIAEKIEKMNLYYL F 13 WP_094346196.1 LynF/TruF/PatF 13 MPLTDFSISNKQVLKFISYHKTSFDIGYIYPLDLFE family peptide O- RFATINKLTMVECSCKVKQNIVYPMRFDISLFGSNY prenyltransferase AQKLNHVMLFFQEINSRVGVNLNLEWLTTFFDRGFK [Nostoc sp. ‘Peltigera FSAILGFNVAIDLRTELKDSRIKLLIGLNNQEYPEK membranacea cyanobiont’ VETALKSCGLESQQGIRTLLIHSSYLVIGFDLYLDG 232] RYDSQLDITLKPDVLQNTEVHQYLLNLISASALRPV PLCNEFAVSFSQMSKDKAIYYDLKNLDDFSTGFSNC LRLSEVSKTIHSIYRSHSIATAMGVVVSDNEFRGDI IQNFNLKYRIYGRT 3 14 AFK79989.1 PirF or putative 14 MIVAEIQKNSLKEQRIKFIRNHQQAFDVEPIYPLRL prenyl transferase FEDFVMSVEGDCSIEASCKIELDKLIASRFMLFFKD [Microcystis  QEWEKYLTQSLAFFRQVENRVGVQLDYSLLQKFLGH aeruginosa PCC  NFDFSKLEVLSAGLDLRTNLADSSLKIHIRIKDYPE 7005] KINQALSLTIDGDDLTAVRDFLSVVGFDFYFDGRSA IEIYPEVKEEDFFKPKTQEKVWQHLPKFVLEPLQVT NLFGFGFSKTNHNPVVYYRLKGRQDLTNYFKINDTA QRVHSFYQHQDILPNMWVGTTQKELEKTRIENIRLY YYKSFKME 15 WP_015081390.1 AcyF or LynF/TruF/ 15 MIANVTQKDRFQEQKLQFIRNHQQAFDVEPIYPLPL PatF family peptide FEDFVMNVEGDCSIEASCKIELDKLIASRFMFFFKD O-prenyltransferase KAQQWQKYLHQSLTFFNRVENLVGVQVDYSLLRQFL [Anabaena sp. 90] GSDFDFRKVTVLSAGIDLRSNIAESSLKMHIRIKDY PEKLDQALSLASNAEDLISVRPFLSLVGFDFYFNGR SEIELYPEIQAEDFAKSETQNLVWRHFPKFVLDPLE VTGSFLVGFSKANPNPVLYYNLKNKQDLANYFKLND AAQRVHSFYQNQDILPHMWVGTVQKELEKTRIENVR LYYYKFFN 16 WP_007355588.1 OscF or LynF/TruF/ 16 MIILSASDSTRPIFTLPKPLTQEQKLHCINAHRQAF PatF family peptide DVQPLYPLDIFQDFITKTDGIDTIEASCKIEADKLQ O-prenyltransferase AARFVALSSQEIERKLTEFLTFFRQVESRVDVQLNY [Kamptonema] DLLHKFLGKSFDFSKVTRITTGVDLRPNISDSSLKI HIRLNDHPENLKKIEAALTLDGNDSTAQRWIALQTV HLIGFDFYLNGRSEIELYCELTEKQFQQPDIQSFLQ QTFPPFVLEPLKVSSVFFTGLSKDNTEPVLYYCLKD KKDLLSYFPINDTAQRVHAFYQNQPVYSSMWVGVAQ GELQKTRIDNIRLYYSKKNYSK

TABLE 4-2B 3 17 WP_039204681.1 LynF/TruF/PatF  17 MIANVTQKDRFQEQKLQFIRNHQQAFDVEPIYPLPL family peptide O- FEDFVMNVEGDCSIEASCKIELDKLIASRFMFFFKD prenyltransferase  KAQQWQKYLHQSLTFFNRVENLVGVQIDYSLLQQFL [Aphanizomenon GSDFDFRKVTVLSAGIDLRSNLAESSLKMHIRIKDY flos-aquae] PEKLDQALSLASNAEDLISVRPFLSLVGFDFYFNGR SEIELYPEIQAEDFAKSETQNLVWRHFPKFVLDPLE VTGSFLVGLSKANPNPVLYYNLKNKQDLANYFKLND AAQRVHSFYQNQDILPK 18 WP_083798475.1 LynF or LynF/TruF/ 18 MTIMAIANRVPYNYLREQRIQFMHAHQDAFDVSTVF PatF family peptide PLPLFEKLVTELEGSNVIELSCKIEADKLLAGRFLI O-prenyltransferase FSDQENNWHQSLAQALQFLDSIESRVGVEINRESLD [Lyngbya sp. PCC  KFLAAHINSGKIMGISTGLDLRPELENSSVKIHIML 8106] GENSEELVRTAIAIDGSHYPVELAQVLLKDTMMIGF DFFLNGHSEVELYISCSRKKDSLPNNRGESTRYYIR QKFSPKVSSLLDASDFFVGGFSKANVEPVLYYAFEN IKDIPKYFVFNDLGNRVYDFCRSQDSITMTWIGINE RDLDRERLNNFRLYYRRSFG 19 ACA04492.1 TruF1 or TruF1 19 MIMTTTWPDSYAKERRIQRLRHHFESFDVERAFPLP [uncultured LFEQAVLSLDSCPLLEPSFKVQEGILFAGRVTTSTG Prochloron sp. TEDWQHLISTALNFFDAVESRVEVTIDRGLLEKFLT 06037A] LHQNSDKIEASLMGIDLRPNVKESSLKVHLRLDPQQ DADELVMTAIDLDGGDYSPELTQVLLKDTFLIGFDF FLDGGSAVEMYTICPGKKPLAMLGKKGAYLKPYVLS NFSHKVTSLLQEVAALTVGFSKENPRPVLYFEFETL REVKYNLLFNSLGDKIYDFCLHNQIENFVSIGVTEP DLEKRRLENFRFYYRKAV 20 AAY21155.1 PatF or PatF 20 MDLIDRLQNNQRKDRRLQFVRTHQEAFDVKPTFPLP [Prochloron  LFEEAILEIEGSCSVESSCQVEGDRLQGGRYEVCNN didemni] QGTTWPESLTHAFKLLDKIDSQLGVRINRDSFDRFA AAHVNSRKIINNTIGVHLGSKLEDSSVMLYINIKPE EDTEELARTALVLDGGRYSDELTRVLLRDTMVIGFE LFFDGRSRVDLGPCAPGKSGTLKMKGKHLEQYTQKN LSRKVNSIFREGYLFGAFFSKTRVEPILFFYHSIIK DLPKYFTFNSLGDKIYNFCQSQGCITDVAIAVTETE LEKSRLENFCFYYDQWDECKPSSDYDTERHLH 21 ACA04493.1 TruF2 or TruF2 21 MVLSQLSKQTNLRENRLRCIRTHLEAFDIEPVLQIS [uncultured  LFEEVIMEVEGSCNVKCSCKVERDRLFACQFTLAYS Prochloron sp. QQKWPKTLKYNAILFDKIKSQVGICIDSSKFEQFSR 06037A] LHVNSDKILDSTVGIDLRPKSQDSCIRISVHLEPKE SPEELVRTALALDNATYTSELTQVFLQDCTAIIFEC FFDGRSRIELGAVAPGKKHGFSGNHGRALTAYAQKY FSPKAVSLSEVSDLFGMTISKYKAEPVLHFGFNNIK DISNYFLFNTLGNRIYSFCQNQDCILLAIIGVNEKE LYSNRLENFLFDYAKNDESRMMRV

Described specifically, the above prenylation enzyme is preferably composed of any of the following amino acid sequences.

(1) An amino acid sequences represented by any of SEQ ID NOS. 1 to 56 listed in the following Table 4-3 to Table 4-8.

(2) An amino acid sequence with one or more amino acid deletions, substitutions or additions in an amino acid sequences represented by any of SEQ ID NOS. 1 to 56.

(3) An amino acid sequence having preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, particularly preferably 98% or more homology with an amino acid sequences represented by any of SEQ ID NOS. 1 to 56.

TABLE 4-3A SEQ Group ID NO: amino acid sequence Enzyme accession No. name or annotation 1 1 MINYANAQLHKSKNLMYMKAHENIFEIEALYPLELFER 1 WP_061432709.1 KgpF or LynF/TruF/ FMQSQTDCSIDCACKIDGDELYPARFSLALYNNQYAEK PatF family peptide QIRETIDFFHQVEGRTEVKLNYQQLQHFLGADFDFSKV O-prenyltransferase IRNLVGVDARRELADSRVKLYIWMNDYPEKMATAMAWC [Microcystis  DDKKELSTLIVNQEFLVGFDFYFDGRTAIELYISLSSE aeruginosa] EFQQTQVWERLAKVVCAPALRLVNDCQAIQIGVSRAND SKIMYYHTLNPNSFIDNLGNEMASRVHAYYRHQPVRSL VVCIPEQELTARSIQRLNMYYCMN 2 MTYPSLLTASQKNLQFIGEHKNAFDLEYLYPLDLFEDL 2 WP_052338757.1 OltF or LynF/TruF/ VAQVEPCRIECSCKIENNKLDPARFNLAINDSFARKAQ PatF family peptide AFLGFFHQVETRVGVIIDYKPLHFFMKGFDYSKVTQIM O-prenyltransferase AGIDARAELTEARVKTWWAIDNYPEKLETAIALSQSDS [Planktothrix] KPLRMLLEINNSLVIGFHLYLRGKSEVRLYPSVSREDF KKVSVQAQLAKILSTPALKLLEDSVAISLVLKENGDKI LHYHVLPERVNSFVNNLHQDMITRAHAPYRNKSQSLGC TVSLSEKELLAGYLKTINLYYMNT 2 3 MITDKHTLVDTNKNLHYIGEHKRAFQVESVYPLDIFER 3 WP_069351383.1 LynF/TruF/PatF FVQEATDWGLECSCKIEKDKLYPIRFNLFRNHPSFKQF family peptide O- DAALDFFQQVEARDDVKLDYKLMHLFLGNYFDFNKIAQ prenyltransferase ILVGVDLRTELSASRLKLWFVIQNYPEKLETAFALCDL [Scytonema millei] KEELRALIVCCSLVVVGFDFYLDGRTNIELYPRILKKE LQEVDVWKQLAKVVSPPTLQLLDSCWAFMLGFSKANPE TILYCPARDPDSFIANLSNDLADRVHAYYQKQPVRGTI VAFRERELLAGAIENLNLYYQMSLGVTKKSEKFDGCVA KTGEAGADIQK 4 MTISENLFIGSQKNLHYINQHKHIFDVDHLYTLNIFDD 4 CEJ46811.1 Anacyclamide FAQKVTHWGLECSCKIQKDQLYPARFNLFRNQPNWQDY synthesis QTAINFFQQVAAKTDVKLTYHLIENFVGNDFDFSKVHQ protein AcyF ILVGIDLRREFSASRLKFWFIISDYPQKLAKAISLCQS [Chrysosporum AEELQPFLVDNSVVIGFDFDFNGGSEIEVYPSISKEKF ovalisporum] QQLAKVLSPQALQLLDSCWSLIIGFSQGNAEKILYYRT SDPNNFIANLRNDLANRVHAYYREQPVQGTIVGLRENE IIAGRIENLNLYYQMSSPITTNKN 5 MTISENLFIGSQKNLHYINQHKHIFDVDHLYTLNIFDD 5 WP_028090374.1 LynF/TruF/PatF FAQKVTHWGLECSCKIQKDQLYPARFNLFRNQPNWQDY family peptide O- QTAINFFQQVAAKTDVKLTYHLIENFVGNDFDFSKVHQ prenyltransferase ILVGIDLRREFSASRLKFWFIISDYPQKLAKAISLCQP [Dolichospermum AEELQPFLVDNSVVIGFDFDFYGGSQIEVYPSISKEKF circinale] QQLAKVLSPQALQLLDSCWSLIIGFSQANPEKILYYRT SDPNNFIANLRNDLANRVHAYYREQPVQGTIVGLRENE IIAGRIENLNLYYQMSSPITTNKN 6 MVTYKPIQVDSEPYLEYIGKHKRAFDIYEDLYPVKLFE 6 WP_099072935.1 LynF/TruF/PatF DFVQVEGRQGGMFRCGCNVTKDELSPARFALLFPTQDD family peptide O- LQSLHNPQQQLEAAFDFFRKVEGRAEVKVNYHLLEKFF prenyltransferase  GKNPDFGGIYFIAVGVDAQTEISESRLKLFIHLHDAPD [Nostoc linckia] KIESAIALSGDSPTLRAFLVNNDLLVGFDFYLDGRSEI EVYPTIYQEELKRADIQSRILPLLPPRALPLLKQCEVL QIGISPANKSNILYFDFVRDPNTFIDNLGNEMAKKVHA YYRHQPVQHVLVGVSEKDLYASSIEKVKLYYFKK

TABLE 4-3B 2  7 MVTYNPIKTNSERYLEYIGKHKLAFDIYEDLYPLKIFE  7 WP_099072934.1 LynF/TruF/PatF DFVEVEAKKRGLFYILSNVDKDEIYPARFCLRFPSLEE family peptide O- AQLLYNPQQQLQTALNFFRQVESRPEVKLNYHHIQQFF prenyltransferase GSISDFQGIILMAVAIDARTVITESRLKLYVWLKNAPE [Nostoc linckia] KVETAIALCGDSPTLRAFLVNDKLQVGFDLFFNGESEI EVYPIISQDELQQVHIRDRIIPLLPPRALPLLQQCAVF QVGFSEANESNILYFDYVHDPNSFVDNLGNEMTKKIHA YYRHQPIKSLTVGIPEHNFYGRAIEHVKLYYDMN  8 MVLKSNKKLYYISANKHAFEIDNLYPLNLFEGFVERIE  8 WP_061432522.1 LynF/TruF/PatF KIEKTENCVLESSCKIDHDKLYPVRFNIGFPNNSIKQL family peptide O- HAVMDFFRRVESRVDVKLNLSLFQQFIGNDFKLDKMTD prenyltransferase LMLGIDLRRDLSDSRLKIGLTIEDYPEKQKAAVILNNN [Microcystis IDEVTSNLLISNRLHIGFDFYLNGRSEMELYPHIMQQD aeruginosa] FQKLDVQQRLSKVLSPPALQVVPACTRICVGISKANRD KIIYYYLENMGDFLNYFTVNDTARKVHAYYLKQPVVEM CVALPESELLAGTTIKNLNLYYLL  9 MTTLLESGKKIYYIGIHKQIFEIKNFYPLDIFDSFVNQ  9 WP_035367793.1 LynF/TruF/PatF IETTSENCSLESSCKIELDKLYPARFGIGFTLKNLKQL family peptide O- NVVYEFFQKVESRIDVQINYSLIQQFFGENFDFNKMTE prenyltransferase FMVGIDARQELSETKLKIALTIKNYPEKIKTAIALNGG [Dolichospermum LDKNIYNLLVSNSLHIGFDLSLDGRSEIELYPYIRNQE circinale] FQIFDIQQRLATVLSPQALQFLPICSRICVGLSKANAD KVVYFYLKNLNDFLNYFTVNDTARRVHAYYQQQPMREM CVAVQEKQLLGGTIEKMNLYYLI 10 MTYPSLLTASQKNLQFIGEHKNAFDLEYLYPLDLFEDL 10 WP_052345004.1 LynF/TruF/PatF VAQVEPCRIECSCKIEDDKLDPARFNLAINDSFARKAQ family peptide O- AFLGFFHQVETRVGVIIDYKPLHFFMKGFDYSKVTQIM prenyltransferase AGIDARAELTEARVKTWWAIDNYPEKLETAIALSQSDS [Planktothrix KPLRMLLEINNSLVIGFHLYLRGKSEVRLYPSVGREDF rubescens] QKISVQAQLAKIISTPALKLLEDSGAISLVLKENGDKI LHYHVLPERVNSFVDNLHQDMITRAHAPYRNKSQSLGC TVSLSEKELIAGSLKTINLYYMNQ 11 MTHPSLLTVSQKNLQFIGEHKNAFDLEYLYPLDLFEDL 11 WP_027254539.1 LynF/TruF/PatF VAQVEPCRIECSCKIENNKLDPARFNLAINDSFARKAQ family peptide O- AFLGFFHQVETRVGVIIDYKPLHFFMKGFDYSKVTQIM prenyltransferase AGIDARAELTEARVKTWWAIDNYPEKLETAIALSQSDS [Planktothrix KPLRMLLEINNSLVIGFHLYLRGKSEVRLYPSVSREDF agardhii] KKVSVQAQLAKILSTPALKLLEDSVAISLVLKKNGDKI LHYHVLPERVNSFVDNLHQDMITRAHAPYRNKSQSLGC TVSLSEKELIAGSLKTINLYYMNQ

TABLE 4-4A SEQ Group ID NO: amino acid sequence Enzyme accession No. name or annotation 2 12 MATSLESSKKNYYISTHKQAFDVENFYP 12 WP_002767616.1 LynF/TruF/PatF  LEIWEKFVEKIEKTSEKCFLESSCKIDQ family peptide  GQFYAARFGIGFDLQNLQQLNAVYNFCQ O-prenyltransferase  EVESRVGVRVDYSLIKQFLGDDFDFSKM [Microcystis TEFLIGVDARRELAESKLKIALTINDYP aeruginosa] EKLKTAIYLNGGLDETIEKLIVSNSLHL GFDLSLNGISEIELYPYIGKQDFQRIDI QQRLATVLSPQALRPLAACRRICVGLSK GNTEKILYYYLEDIKDFLNYFTPNDTAR RVHAYYQKQPISEMCVAAPESQFIAEKI EKMNLYYLF 13 MPLTDFSISNKQVLKFISYNKTSFDIGY 13 WP_094346196.1 LynF/TruF/PatF  IYPLDLFERFATINKLTMVECSCKVKQN family peptide  IVYPMRFDISLFGSNYAQKLNHVMLFFQ O-prenyltransferase  EINSRVGVNLNLEWLTTFFDRGFKFSAI [Nostoc sp. LGFNVAIDLRTELKDSRIKLLIGLNNQE ‘Peltigera YPEKVETALKSCGLESQQGIRTLLIHSS membranacea YLVIGFDLYLDGRYDSQLDITLKPDVLQ cyanobiont’ 232] NTEVHQYLLNLISASALRPVPLCNEFAV SFSQMSKDKAIYYDLKNLDDFSTGFSNC LRLSEVSKTIHSIYRSHSIATAMGVVVS DNEFRGDIIQNFNLKYRIYGRT 3 14 MIVAEIQKNSLKEQRIKFIRNHQQAFDV 14 AFK79989.1 PirF or putative EPIYPLRLFEDFVMSVEGDCSIEASCKI prenyl transferase ELDKLIASRFMLFFKDQEWEKYLTQSLA [Microcystis  FFRQVENRVGVOLDYSLLQKFLGHNFDF aeruginosa PCC SKLEVLSAGLDLRTNLADSSLKIHIRIK 7005] DYPEKINQALSLTIDGDDLTAVRDFLSV VGFDFYFDGRSAIEIYPEVKEEDFFKPK TQEKVWQHLPKFVLEPLQVTNLFGFGFS KTNHNPVVYYRLKGRQDLTNYFKINDTA QRVHSFYQHQDILPNMWVGTTQKELEKT RIENIRLYYYKSFKME 15 MIANVTQKDRFQEQKLQFIRNHQQAFDV 15 WP_015081390.1 AcyF or LynF/TruF/ EPIYPLPLFEDFVMNVEGDCSIEASCKI PatF family ELDKLIASRFMFFFKDKAQQWQKYLHQS peptide O- LTFFNRVENLVGVQVDYSLLRQFLGSDF prenyltransferase DFRKVTVLSAGIDLRSNIAESSLKMHIR [Anabaena sp. 90] IKDYPEKLDQALSLASNAEDLISVRPFL SLVGFDFYFNGRSEIELYPEIQAEDFAK SETQNLVWRHFPKFVLDPLEVTGSFLVG FSKANPNPVLYYNLKNKQDLANYFKLND AAQRVHSFYQNQDILPHMWVGTVQKELE KTRIENVRLYYYKFFN 16 MIILSASDSTRPIFTLPKPLTQEQKLHC 16 WP_007355588.1 OscF or LynF/TruF/ INAHRQAFDVQPLYPLDIFQDFITKTDG PatF family IDTIEASCKIEADKLQAARFVALSSQEI peptide O- ERKLTEFLTFFRQVESRVDVQLNYDLLH prenyltransferase KFLGKSFDFSKVTRITTGVDLRPNISDS [Kamptonema] SLKIHIRLNDHPENLKKIEAALTLDGND STAQRWIALQTVHLIGFDFYLNGRSEIE LYCELTEKQFQQPDIQSFLQQTFPPFVL EPLKVSSVFFTGLSKDNTEPVLYYCLKD KKDLLSYFPINDTAQRVHAFYQNQPVYS SMWVGVAQGELQKTRIDNIRLYYSKKNY SK

TABLE 4-4B 3 17 MIANVTQKDRFQEQKLQFIRNHQQAFDVEPIYPLP 17 WP_039204681.1 LynF/TruF/PatF LFEDFVMNVEGDCSIEASCKIELDKLIASRFMFFF family peptide KDKAQQWQKYLHQSLTFFNRVENLVGVQIDYSLLQ O-prenyltransferase QFLGSDFDFRKVTVLSAGIDLRSNLAESSLKMHIR [Aphanizomenon IKDYPEKLDQALSLASNAEDLISVRPFLSLVGFDF flos-aquae] YFNGRSEIELYPEIQAEDFAKSETQNLVWRHFPKF VLDPLEVTGSFLVGLSKANPNPVLYYNLKNKQDLA NYFKLNDAAQRVHSFYQNQDILPK 18 MTIMAIANRVPYNYLREQRIQFMHAHQDAFDVSTV 18 WP_083798475.1 LynF or LynF/TruF/ FPLPLFEKLVTELEGSNVIELSCKIEADKLLAGRF PatF family peptide LIFSDQENNWHQSLAQALQFLDSIESRVGVEINRE O-prenyltransferase SLDKFLAAHINSGKIMGISTGLDLRPELENSSVKI [Lyngbya sp.  HIMLGENSEELVRTAIAIDGSHYPVELAQVLLKDT PCC 8106] MMIGFDFFLNGHSEVELYISCSRKKDSLPNNRGES TRYYIRQKFSPKVSSLLDASDFFVGGFSKANVEPV LYYAFENIKDIPKYFVFNDLGNRVYDFCRSQDSIT MTWIGINERDLDRERLNNFRLYYRRSFG 19 MIMTTTWPDSYAKERRIQRLRHHFESFDVERAFPL 19 ACA04492.1 TruF1 or TruF1 PLFEQAVLSLDSCPLLEPSFKVQEGILFAGRVTTS [uncultured TGTEDWQHLISTALNFFDAVESRVEVTIDRGLLEK Prochloron FLTLHQNSDKIEASLMGIDLRPNVKESSLKVHLRL sp. 06037A] DPQQDADELVMTAIDLDGGDYSPELTQVLLKDTFL IGFDFFLDGGSAVEMYTICPGKKPLAMLGKKGAYL KPYVLSNFSHKVTSLLQEVAALTVGFSKENPRPVL YFEFETLREVKYNLLFNSLGDKIYDFCLHNQIENF VSIGVTEPDLEKRRLENFRFYYRKAV 20 MDLIDRLQNNQRKDRRLQFVRTHQEAFDVKPTFPL 20 AAY21155.1 PatF or PatF PLFEEAILEIEGSCSVESSCQVEGDRLQGGRYEVC [Prochloron NNQGTTWPESLTHAFKLLDKIDSQLGVRINRDSFD didemni] RFAAAHVNSRKIINNTIGVHLGSKLEDSSVMLYIN IKPEEDTEELARTALVLDGGRYSDELTRVLLRDTM VIGFELFFDGRSRVDLGPCAPGKSGTLKMKGKHLE QYTQKNLSRKVNSIFREGYLFGAFFSKTRVEPILF FYHSIIKDLPKYFTFNSLGDKIYNFCQSQGCITDV AIAVTETELEKSRLENFCFYYDQWDECKPSSDYDT ERHLH 21 MVLSQLSKQTNLRENRLRCIRTHLEAFDIEPVLQI 21 ACA04493.1 TruF2 or TruF2 SLFEEVIMEVEGSCNVKCSCKVERDRLFACQFTLA [uncultured YSQQKWPKTLKYNAILFDKIKSQVGICIDSSKFEQ Prochloron sp. FSRLHVNSDKILDSTVGIDLRPKSQDSCIRISVHL 06037A] EPKESPEELVRTALALDNATYTSELTQVFLQDCTA IIFECFFDGRSRIELGAVAPGKKHGFSGNHGRALT AYAQKYFSPKAVSLSEVSDLFGMTISKYKAEPVLH FGFNNIKDISNYFLFNTLGNRIYSFCQNQDCILLA IIGVNEKELYSNRLENFLFDYAKNDESRMMRV

TABLE 4-5A SEQ Group ID NO: amino acid sequence Enzyme accession No. name or annotation 4 22 MNCTSILQQNHLREKRLQFIRAHQTAFEVEPVFPLQ 22 WP_008049959.1 LynF/TruF/PatF  LFEDFVVSVEGDCTIEASCKVESDHLIASRFLLFFQ family peptide O- EMTQSWPQKLDQAFRFFHQTENQVDVHLDYGLLQHF prenyltransferase  LGNDFDFSKMSVLSTGIDLRQNLADSSLKMHITIED [Arthrospira sp. YPEKIATAFSLAKLPRDNFHQILLSSVSLIGFDFYL PCC 8005] DGRSEIELYASLKEEEFNSPHVQSFLASIFCASALK PLGASSSFYMGLSIANENPVLYYLLKNKQELQNYFR LNDMGNRVHSFYNQQEILPHAWVGVAEQELQQGRIE NIRLYYYQRFAAGMTG 23 MIADVIQKDRIKEQKLQFIRNHQQAFDVEPIYPLPL 23 WP_042155601.1 LynF/TruF/PatF  FEDFVMGVEGDCSIEASCKIELDKLIASRFLLLFFK family peptide O- DKAQQWQKYLHQSLTFFSQVENRVGVQIDYSLLQQF prenyltransferase  LGSNFDFSKVRTFTTGIDLRNNLADSSLKIHFRIED [Planktothrix YPEKLETSLALSGDSISSLIKELPSYPLTKLIPQIG agardhii] FDFYFNGSNEIELYIEVGEDDFQHPEIKEFLWQRFS KIALEPLKASSLFHLGLSKANNDPVLYYHLKNKKDL VNYFNLNDMGQRVHSFYQHQDVGVPMWVGTAQKELE KTRIENIRLYYYKSFKMESN 24 MIADVIQKDRLKEQKLQFIRNHQQAFDVEPIYPLPL 24 BBD55900.1 hypothetical  FEDFVMGVEGDCSIEVSCKIESDKLIASRFLLFFKD protein KTQQWQKYLHQSLTFFSQVENRVGVQIDYSLLQQFL NIES204_32190  GSNFDFSKVRTFTTGIDLRNNLADSSLKIHFRIEDY [Planktothrix PEKLETSLALSGDSISSLIKELPSYPLTKLIPQIGF agardhii NIES-204] DFYFNGSNEIELYIEVGEDDFQHPEIKEFLWQRFSK IALEPLKASSLFHLGLSKANNDPVLYYHLKNKKDLV NYFNLNDMGQRVHSFYQHQDVGVPMWVGTAQKELEK TRIENIRLYYYKSFKMESN 25 MIVADIQKSFLKEQRLQFIRNHQQAFDVELIYPLRL 25 REJ40736.1 LynF/TruF/PatF  FEDFVMGVEGDCSIEASCKIELDKLIASRFMLFFKD family peptide O- KAQEWQKYLAQSLAFFRQVENRVGVKLDYSLLQQFL prenyltransferase  GLNFNFSKITVFSTGIDLRTNLADSSLKMHIRLKDY [Microcystis  PEKINQALLLTSDSDDLIAVRDFLSIVGFDFYFDGR flos-aquae DF17] SAIKIYPEVAETDFFKPETQDKVWRHLPKFVLEPLK ATSLFCFGFSKTNNNPVLYYRLKNRQDLTNYFKLND TAQRVHSFYQHQDILSSVWVGTAQQELEKTRIENVR LYYYKYFGME 26 MIVADIQKSSLKEQRLQFIRNHQQAFDVELIYPLRL 26 WP_002797466.1 LynF/TruF/PatF  FEDFVMGVEGDCSIEASCKIELDKLIASRFMLFFKD family peptide O- KAQEWQKYLAQSLAFFQQVENRVGVKLDYSLLQQFL prenyltransferase  GLNFNFSKITVFSTGIDLRTNLADSSLKMHIRLKDY [Microcystis PEKINQALLLTSDSDDLIAVRDFLSVVGFDFYFDGR aeruginosa] SAIKIYPEVAETDFFKPETQDKVWRHLPKFVLEPLK ATSLFGFGFSKTNNNPVLYYRLKNRQDLTNYFKLND TAQRVHSFYQHQDILPNMWVGTSQQELEKTRIENVR LYYYKYFGME

TABLE 4-5B 4 27 MIVAEIQKNSLKEQRIKFIRNHQQAFDVEPIYPLRL 27 WP_024969547.1 LynF/TruF/PatF FEDFVMSVEGDCSIEASCKIELDKLIASRFMLFFKD family peptide O- QEWEKYLTQSLAFFRQVENRVGVQLDYSLLQKFLGH prenyltransferase NFDFSKLEVLSAGLDLRTNLADSSLKIHIRIKDYPE [Microcystis KINQALSLTIDGDDLIAVRDFLSVVGFDFYFDGRSA aeruginosa] IEIYPEVKEEDFFKPKTQEKVWQHLPKFVLEPLQVT NLFGFGFSKTNHNPVVYYRLKGRQDLTNYFKINDTA QRVHSFYQHQDILPNMWVGTTQKELEKTRIENIRLY YYKSFKME 28 MIVADIQQNSLKEQRTQFIRNHQQAFDVEPVYPLRL 28 REJ40476.1 LynF/TruF/PatF FEDFVMGVEGDCTIEASCKIELDKLIASRFLLFFKD family peptide O- KAQEWQKYLAQSLAFFQQVENRVGVQLDYSLLQRFL prenyltransferase GDNFDFSKLEVLSAGIDLRTNLADSSVKMHIRIGDY [Microcystis flos- PEKLATAFVLSDGVADSNYLSGFVHLIGFDFYFNGK aquae TF09] SAIEIYAEVTEDDFFKPEIINQVWQHFPKSALKPLQ ASSLFFTGLSKANHNPVLYYHLKNKQDLANYFKLND TAQRVHSFYQHQDILPNMWVGTAQQELEKTRIENVR LYYYKYFGME 29 MIVADIQQNSLKEQRTQFIRNHQQAFDVEPVYPLRL 29 WP_002792928.1 LynF/TruF/PatF FEDFVMGVEGDCTIEASCKIELDKLIASRFLLFFKD family peptide O- KAQEWQKYLAQSLAFFQQVENRVGVQLDYSLLQRFL prenyltransferase GDNFDFSKLEVLSAGIDLRTNLADSSVKMHIRIGDY [Microcystis PEKLATAFVLSDGVADSNYLSGFVHLIGFDFYFNGK aeruginosa] SAIEIYAEVTEDDFFKPEIINQVWQHFPKSALKPLQ ASSLFFTGLSKANHNPVLYYHLKNKQDLANYFKLND TAQRVHSFYQHQDILPNMWVGTAQQELEKTRIENVR LYYYKYFGMD 30 MIVADIQKSSLKEQRLQFIRNHQQAFDVEPIYPLRL 30 WP_004161245.1 LynF/TruF/PatF FEDFVMGVEGDCSIEASCKIELDKLIASRFMLFFKD family peptide O- KAQQWQKYLDQSLAFFQQVENRVGVQLDYSLLQRFL prenyltransferase GDNFDFSKLEVLSAGIDLRTNLADSSVKMHIRIGDY [Microcystis PEKLATAFVLSDGAADSNHLSGFVNLIGFDFYFNGK aeruginosa] SEIEIYAEVTEDDFFKPETINQVWQHFPKSALKPLQ ASSLFFTGLSKANHNPVLYYSLKNCQDLINYFKLSD TAQRVHSFYQHQDILPSMWVGTAQQELEKTRIENVR LYYYKYFGME 31 MIVADIQKSSLKEQRLQFIRNHQQAFDVEPIYPLRL 31 WP_012263813.1 LynF/TruF/PatF FEDFVMGVEGDCTIQASCKIELDQLIASRFMLFFKD family peptide O- KAQEWQNYLAQSLAFFRQVENRVGVKLDYSLLQQFL prenyltransferase GLNFNFSKITVFSTGIDLRTNLADSSLKMHIRLKDY [Microcystis PEKINQALLLTSDSDDLIAVRDFLSIVGFDFYFDGR aeruginosa] SAIKIYPEVAETDFFKPETQDKVWRHLPKFVLEPLK ATSLFGFGFSKTNNNPVLYYRLKNRQDLTNYFKLND TAQRVHSFYQHQDILSSVWLGTAQQELEKTRIENVR LYYYKLFGKL

TABLE 4-6A SEQ Group ID NO: amino acid sequence Enzyme accession No. name or annotation 4 32 MIADVIQKDRVKEQKLQFIRNHQQAFDVEPIYPL 32 WP_026786030.1 LynF/TruF/PatF PLFEDFVMGVEGDCSIEASCKIESDKLVASRFMF family peptide O- FFQDKAQQWQKYLNQSLTFFSQVENRVGVQIDYS prenyltransferase LLQQFLGSNFDFSKVTVLSAGIDLRDNLAESSLK [Planktothrix MHIRIKDYPEKLNKALFLTSDSEDLISVRRYLSV rubescens] VGFDFYFNGRSEIELYPEVQEEDFAKPEIQDLVW RHFPKFVLEPLKVTSLFGFGFSKANNNPVLYYHL KNRQDLTNYFKLNDTAQRVHSFYQHQDRLPNMWI GTAQKELEKTRIENIRLYYYKSFKMESS 33 MIADVIQKDRVKEQKLQFIRNHQQAFDVEPIYPL 33 WP_027249705.1 LynF/TruF/PatF PLFEDFVMGVEGDCSIEASCKIESDKLVASRFMF family peptide O- FFQDKAQQWQKYLNQSLTFFSQVENRVGVQIDYS prenyltransferase LLQQFLGSNFDFSKVTVLSAGIDLRDNLAESSLK [Planktothrix MHIRIKDYPEKLNKALFLTSDSEDLISVRRYLSV agardhii] VGFDFYFNGRSEIELYPEVQEEDFAKPEIQDLVW RHFPKFVLEPLKVTSLFGFGFSKANNNPVLYYHL KNRQDLTNYFKLNDTAQRVHSFYQHQDRLPNMWI GTAQKELEKTRIENIRLYYYKSFKMESN 34 MIVADIQKSSLKEQRLQFIRNHQQAFDVEPIYPL 34 WP_002762729.1 LynF/TruF/PatF RLFEDFVMGVEGDCTIEASCKIELDKLIASRFLL family peptide O- FFKDKAQEWQKYLAQSLAFFQQVENRVGVQLDYS prenyltransferase LLQGFLGDNFDFSKLEVLTAGIDLRTNLADSSLK [Microcystis MHIRIGDYPEKLATAFVLSDGVADSNYLSGFVHL aeruginosa] IGFDFYFNGKSAIEIYAEIREDDFFKPETINQVW QHFPKSALKPLQASDLFFTGLSKANNNPVLYYSL KNRQNLINYFKLNDTAQRVHSFCQHQDILPNMWV GTAQQELEKTRIENVRLYYYKYFGME 35 MIADVIQKDRIKGQKLQFIRNHQQAFDVEPIYPL 35 WP_027254541.1 LynF/TruF/PatF PLFEDFVMGVEGDCSLEVSCKIESDKLIASRFLL family peptide O- FFKDKTQQWQKYLNQSLTFFSQVENRVGVQIDYS prenyltransferase LLQQFLGYNFDFSKVTVLSAGIDLRNNLAESSLK [Planktothrix MHIRIKDYPEKVETAFSLSDGAADGNYLKDFVNL agardhii] IGFDFYFNGKSEIEIYAEVQEEDFFKPETINLVW RHFPPSVLTSLQASDSFFVGLSKPNNNPVLYYHL KNRQDLVNYFKLNDTAQRVHSFYQHQDILPEMWV GTAQKELEKTRIENIRLYYYKSFKMESN 36 MNCTSVLQQNHLREKRLQFIRAHQTAFDVEPVFP 36 WP_006617852.1 LynF/TruF/PatF LQVFEDFVFGVEGDCTIEASCKVESDHLIASRFL family peptide O- LFFQEMTQSWPQKLDQAFRFFHQTENQVGVRLDY prenyltransferase GLLQHFLGDDFDFSKISVLSTGIDLRQNLADSSL [Arthrospira KMHITIEDYPEKIATAFSLAKLPRDKFHQILLSS platensis] VSLIGFDFYLDGRSEIELYASLKEEEFNSPHVQS FLTSNFCASALKPLAASSAFYMGLSIANENPVLY YLLKNKQELQNYFRLNDTGNRVHSLL

TABLE 4-6B 4 37 MIVAEIQKNSLKEQRLQFIRNHQQAFDVEPIYPLR 37 WP_002792921.1 LynF/TruF/PatF LFEDFVVGVEGDCSIEASCKIELDKLIASRFMLFF family peptide O- KDKAQEWQKYLAQSLAFFKQVENRVGVKLDYSLLQ prenyltransferase QFLGTHFDFSKLQEVLSAGLDLRSNLADSSLKIHI [Microcystis RIKDYPEKLETAFALSDGAADSNYLSGFVNLIGFD aeruginosa] FYFNGKSEIEIYAELQEEDFFKPETINLVWRHFPH SVLKPLQASNLFFTGLSKANHDPVLYYHLKDRQDL ANYFNLNDTAQRVHSFYQHQDILPTMWVGTSQQQL EKTRIENVRLYYYKFFGKLESISC 38 MPREQRLQFIKAHQAAFEVEPLYPLALFEALVETF 38 WP_012626000.1 LynF/TruF/PatF DEDCALEASCKIEFDQLIASRFLIFFSQNFEQNLA family peptide O- RVLNFMTQVNQRVDVQINTDLLYHFLGQKFDFRKM prenyltransferase IRLATGVDLRSNLADSSLKIHIRLEDYPEKIESAL [Cyanothece sp. ALHGNPDDASYWADLNAIANIGLDFYLDGRSEIEF PCC 7425] YPELSEERFQQPEMQVLLQQMFPPFVLAPLKASEI FGFGLSKANPSAVLYYQLKNKQDLPSYFAINDKAH QVHGYYLHQDTRPYMCVGVAQSELAKTRIDQIRLY YHQFFKVQNP 39 MIANVTQKDRFKEQKLQFIRNHQQAFDVEPIYPLP 39 OBQ18952.1 anacyclamide LFEDFVMNVEGDCSIEASCKIELDKLIASRFMFFF synthesis protein KDKAQQWQKYLHQSLTFFNRVENLVGVQIDYSLLQ AcyF [Anabaena QFLGSDFDFRKVTVLSAGIDLRSNLAESSLKMHIR sp. AL93] IKDYPKKLDQALSLASNAEDLISVRRFLSVVGFDF YFNGRSEIELYPEVQEEDFAKSETQNLVWRHFPKF VLDPLEVTRLFGFGLSKANHNPVLYYNLKNKQDLA NYFKLNDAAQRVHSFYQNQDILPNMWVGTVQKELE KTRIENVRLYYYKSFN 40 MIVGAVPKERIKEQKLQFIRNHQQAFDVEPIYPLP 40 PZV25905.1 anacyclamide LFEDFVMSVEGDCTIEASCKVELDKLIASRFLFFF synthesis protein KDKAQDWPSYLSQGLSFFKQVETQVGVQLDYSLLQ AcyF [Snowella SFLGTDFDFSRVRTFTTGIDLRRESLAESSVKIHF sp.] RVKDYIEKVQTAIILSGGEFGFLEDFPKKFLAQYI PQIGFDFYFDGRCEIELYFEVIECNFGDPNVRQYL WSKMPSIALAPLKDTAVFHLGLSKANTNPVFYYQL KKYKGNLLNYFRLNDAAMRVDSFYRNQDTEYGGWV GVALEELEKTRIENIRLYYHKYFKIE 41 MIVADIQKSSLKEQRLQFIRNHQQAFDVEPIYPLR 41 WP_002802539.1 LynF/TruF/PatF LFEDFVMGVEGDCTIEASCKIELDKLIASRFLFCF family peptide O- KDPEQHWPSYLSQGLSFFKQVENRVGVQFDYSLLQ prenyltransferase AFLGTDFDFSHVTTFTTGIDLRRESLTESSVKIHF [Microcystis RIEDYIAKVQTAIILSGYEVGFLEDFPKQFLARYI aeruginosa] PQIGFDFYFDGRCEIELYFEVKENNFDDPNVKKYL WSKMPPVALAPLKDTDVFHLGLSKANTNPVFYYQL KKYKGDLLNHFRLNDTAMRVDSFYRNQETDYGGWV GVALEELEKTRIENIRLYYHKYFGME

TABLE 4-7A SEQ Group ID NO: amino acid sequence Enzyme accession No. name or annotation 4 42 MIADVIQKDRLKEQKLQFIRNHQQAFDVEPIYPLP 42 WP_079678526.1 LynF/TruF/PatF LFEDFVMSVDGDCTIEASCKVELDKLIASRFLFFF family peptide O- KDKAQDWPSYLSQGLSFFKQVETQVGVQLDYSLLQ prenyltransferase AFLGTDFDFSRVRTFTTGIDLRRESLAESSVKIHF [Planktothrix sp. RIEDYIEKVQTAIILSGGEFGFLEDFPKKFLARYI PCC 11201] PQIGFDFYFNGRCEIELYFEVIECNFDDPNVKQYL WSKMPPIALAPLKDTAVFHLGLSKANTNPVFYYQL KKYKGNLLNYFRLNDDAMRVDSFYRNQDTDYGGWV GVALEELEKTRIENIRLYYHKYFKMESN 43 MAKLSSAVFSFFRQVENRVGVQLDYSLLQQFLGDN 43 CCH91680.1 conserved FDFSKLEVLSTGIDLRTNLADSSVKMHIRIKDYPE hypothetical KLETAFLLSDGAAGSNYLSGFVNLIGFDFYFNGKS protein EIEIYAEVGEDDFFKPETINQVWRHFPDSVLKPLQ [Microcystis  ASSLFFTGLSKANNNPVLYYYLKNRQDLINYFRLN aeruginosa PCC DTAQRVHSFYEHQDILPYMWVGTAQQELEKTRIEN 9432] VRLYYYKLFGKLE 44 MVILTPTSRTNRSQEQRLQFIRAHQNAFDVDSNFL 44 WP_044449669.1 LynF/TruF/PatF LSTFEKTVLEIDENCGIEPSCKIEGDHLFAGRYAV family peptide O- AGEDGGNWRKILAQSLGFLNTIESRVDFKINRELI prenyltransferase QKFLALHIGSNKIIANTTGIDLRPSLEESSVKIHF [Mastigocladus KIKVDEDAEELARTAIALDGGYYSSELTQVLLKDT laminosus] TLIGFDFFLNGHSEVELYPSCPGGKYELSSNWGRT LSTYVQQNFSKKVVSLFNVCDIFMAGFSKVNIEPV LYFVFFDIKDIAKYFLFNSLGDRIYNFCQSQDDDY SSYYCVGVNERDLRVNRLEKFRFYYTKVI 45 MTFTPAFQNSILQERRLRFMRSHQEAFDVEPEFPL 45 WP_045871562.1 LynF/TruF/PatF PLLENLAQALGSRCVVELSCKVESNQLFAARINIC family peptide O- YRDAWPQSLGQSLKFLDEVESRVGIQINRDLLQQF prenyltransferase LAVHINSNKILWNTTGIDLRPNVEDSSVKIHIGID [Nostocales] PNQDTEELVMTAIGLDGSQYSPELIQVLLKDSYMI GFDFFLNGRSEVELYTSCPGGKQQLVGNQGIYLKS YAKRNFSEKVFYLLEACDLFMAGFSKANTEAVIYF GFNNIEDMPKYFLFTSLGQRIYDFCRSQGAGPLPC VGVTQKDLESHHVENLRFYYRREFS 46 MTSSLLEVLSAGIDLRTDLADSSVKMHIRIGDYPE 46 WP_002758021.1 LynF/TruF/PatF KLATAFILSDGAADSNYLSGFVNLIGFDFYFNGKS family peptide O- EIEIYVEVREDDFFKPETINQVWQHFPKSALKPLQ prenyltransferase SSSLFFTGLSKANHNPVLYYNLKNPQDLINCFKLN [Microcystis YTAQKVHSFYQHQDILPNMWVGTAQQELEKTRIEN aeruginosa] IRLYYYKSFTME

TABLE 4-7B 4 47 MAIANRVPYNYLREQRIQFMHAHQDAFDVSTVFPLP 47 EAW34319.1 hypothetical LFEKLVTELEGSNVIELSCKIEADKLLAGRFLIFSD protein QENNWFIQSLAQALQFLDSIESRVGVEINRESLDKF L8106_29075 LAAHINSGKIMGISTGLDLRPELENSSVKIHIMLGE [Lyngbya sp.  NSEELVRTAIAIDGSHYPVELAQVLLKDTMMIGFDF PCC 8106] FLNGHSEVELYISCSRKKDSLPNNRGESTRYYIRQK FSPKVSSLLDASDFFVGGFSKANVEPVLYYAFENIK DIPKYFVFNDLGNRVYDFCRSQDSITMTWIGINERD LDRERLNNFRLYYRRSFG 48 MTSSKLEVLSAGIDLRTDLADSSVKMHIRIGYYPEK 48 WP_004161274.1 LynF/TruF/PatF LATAFILSDGAADSNYLSGFVNLIGFDFYFNGKSEI family peptide O- EIYAEVREDDFFKPETINQVWQHFPKSALKPLQASS prenyltransferase LFFTGLSKANHNPVLYYNLKNPQVLINCFKLNYTAQ [Microcystis KVHSFYQHQDILPNMCVGTAQQELEKTRIENIRLYY aeruginosa] YKSFTME 49 MTSSLLEVLSAGIDLRTNLADSSVKMHIRIGDYTEK 49 WP_002769120.1 LynF/TruF/PatF LATAFILSDGAADSNYLSGFVNLIGFDFYFNGKSEI family peptide O- EIYAEVREDDFFKPETINQVWQHFPKSALKPLQASS prenyltransferase LFFTGLSKANHNPVLYYNLKNRQDLTNYFKINDTAQ [Microcystis RVHSFYQHQDILPKMWVGTAQQELEKTRIENVRLYY aeruginosa] YKYFGME 50 MILTTTWQDNYAKEQCIQYLRHHFESFEVEPAFPLP 50 RAM49414.1 microcyclamide LFEQAILKLNSCSVLEPSFKVQGSTLFAGRVTASTK biosynthesis AASTKKDWPLLIRSALNLFDAVESRVEATINRSLLE protein KFLMLHQDSGKIEGSLMGIDLRPNVKESSLKVHLHL [Hapalosiphonaceae DKEQAVDELVMTAINLDGGYYAPDLTQVLLKDTALI cyanobacterium JJU2] GFDFFLDGRSAVEMYTCCAGSKQLSFLGKRGAYIKP YIYKNFSQKVTSLLKDVALVMVGFSKENSKPVLYFE FEKLRDIKSHLLFNSLGDKIYDFCLHHQLENFVSIG VTEQDLEKSRLENFRFYYRKSV 51 MTITSGFSHNYLREQRLRFIRTHLDAFDVEPCFPLP 51 WP_054466071.1 LynF/TruF/PatF LFEEAVLEIEGYCGIEPTCHVEGDRLFASDFQVATY family peptide O- EETWPRSLVDASKFLDKVESRVGVQINRNLLDQFSS prenyltransferase LYINSSQIETNTIGIDLRPTLQDSLIKVYMHLHPGE [Planktothricoides GNEDLVMTALALDGANYSAEVTEVLLKDGSLIGFNL sp. SR001] FLDGRSNIEIWAISPGGKYQQRGNFGSYLAAYIRKH FSQKVISLFDVSDGIAASFSKQKTSPLLYFHFFDIK EIPKYFAFNSLGDKIYDFYQSLDCVTYATVYAREQD MESNRLNNYGFLYNRSDTCQVDLDKLRFTQQVGISP 52 MTITPVFSHHYLREQRLRFIRTHLEAFDVEPRFPLP 52 WP_002754527.1 LynF/TruF/PatF LFEEAVLEIEGYCGIEPTCHVQGDRIFAGDIQVATY family peptide O- EKTWPKSLMGASKFLDKVGSQVGVQINRNLLDRFSS prenyltransferase LYIHSSKIETNTIGIDLRPTIQDSLIKIYMHLHPGG [Microcystis SHEDLVMTALALDGANYSAEVTEVLLKDGALIGFNL aeruginosa] FLDGRSNIEIWAISPGGKYQHKGNFGRDLAAYIRKN FSQKVNFLFDVADGIAASFSKQKIDPIFYFHFFDIK DIPKYFAFNSLGDRIYDFYQSQDCVTYASISVREQD LESSRLDNYGFLYNKSDVCQLNLDKFHFAQQLGISP

TABLE 4-8 SEQ ID accession name or Group NO: amino acid sequence Enzyme No. annotation 4 53 MPITSVFSHNYLREQRLRFIRTHRDAFDVPYSFLLPLYEEAVFK 53 WP_015177266.1 LynF/TruF/PatF IEGSCGVEPSCNVEGDRLFAGDFQVANYGHTWPRSLIDAASFLD family KIERRIDVKINRNLLERFSALYIGSSKIENNTIGIDLRPTLQDS peptide O- VIKIYMHIHPGQSNEDLVMTALALDGAIYSAELTQVLLRDVVVI prenyltransferase GFNLFLDGRSNIEIWAGSAGQKHNHQGNLGRDLTAYIRKYYSHK [Oscillatoria VNYMFNVSDFSAVSFSKQKIDPLFHFHFFDIKDILKYFAFNSLG nigro-viridis] DKIYDFCQSQDCITYTGVSARAQELESNRLNNYSFFYNQSDTCQ TDLDILRLPMMF 54 MLQNIQNNRFKDRPIEFIRSHQEAFDVEPIFPIPLFEEVILELE 54 CUR21328.1 PatF GVFSVKPSCKVEGSRLLAGRYETGILPMRKTWPQLLTHTFKFLN [Planktothrix KIESRVGVCINRESFEQFAAVHIGSDKIMDSTIGVHLGSKLEDS paucivesiculata SVMMYIHFDTEQDCEELAKTAIALDGGHYSDELTQVLIRDMVVI PCC 9631] GFELFFDGRSHVELCPVAPAQPGALGIRGKYLTPYIQKYFSQKV NSIFRGSHLLKICFSKAYATPVLGFYFKNLKDIPKYFRFNSLGD RIYDFCQSQSCIMVPAVFATEAELEKSRLEKFDFYYIRWEEC 55 MIVNVIQKDRLKEQKLQFIRNHQQAFDVEPIYPLPLFEDFVTSI 55 5TTY_A PagF Chain A, EGDCSLEASCKIESDKLIASRFLLFFEDKTQEWQKYLHQSLTFF Pagf GLVENRVGVKINYSLLQQFLGSSFDFSKVTVLSAGIDLRNNLAE Prenyltransferase SSLKMHIRIKDYPEKLDKAFALSDGAADGNYLKDFVNLIGFDFY FNGKSEIEIYAEVQEDDFFKPEINNLVWQHFPKTALQPLKASSL FFTGLSKANNNPVLYYHLKNRQDLTNYFKLNDTAQRVHSFYQHQ DILPYMWVGTAQKELEKTRIENIRLYYYKSFKMESN 56 DLIDRLQNNQRKDRRLQFVRTHQEAFDVKPTFPLPLFEEAILEI 56 4BG2_A PatF Chain A, EGSCSVESSCQVEGDRLQGGRYEVCNNQGTTWPESLTHAFKLLD X-ray Crystal KIDSQLGVRINRDSFDRFAAAHVNSRKIINNTIGVHLGSKLEDS Structure Of Patf SVXLYIHIKPEEDTEELARTALVLDGGRYSDELTRVLLRDTXVI From Prochloron GFELFFDGRSRVDLGPCAPGKSGTLKXKGKHLEQYTQKNLSRKV Didemni NSIFREGYLFGAFFSKTRVEPILFFYHSIIKDLPKYFTFNSLGD KIYNFCQSQGCITDVAIAVTETELEKSRLENFCFYYDQWDECKP SSDYDTERHLHRS

The term “with one or more amino acid deletions, substitutions or additions” as used herein means that the number of amino acids deleted, substituted or the like is not particularly limited insofar as a prenylation enzyme thus obtained retains a function of prenylating an amino acid residue. In this sentence, the term “more” means an integer of 2 or more, preferably several, for example, from 2 to 15, more preferably from 2 to 10, still more preferably from 2 to 5, still more preferably 2, 3, or 4. The position of deletion, substitution or addition in each prenylation enzyme may be any of N terminal, C terminal and therebetween in each prenylation enzyme insofar as the resulting prenylation enzyme retains a function of prenylating an amino acid residue.

The term “having Y % or more homology with an amino acid sequence represented by SEQ ID NO: X” as used herein means that when respective amino acid sequences of two polypeptides are aligned to have the maximum agreement, a proportion of the number of amino acid residues common to them to the total number of amino acids shown by SEQ ID NO: X is Y % or more.

The prenylation enzyme is capable of prenylating a peptide containing a motif of, for example but not particularly limited to, X2X1X3 or X4X2X1X3X5 (X1 represents W which is an amino acid residue or derivative thereof to be prenylated, X2, X3, X4, and X5 each independently represent an arbitrary amino acid residue. The arbitrary amino acid residue also includes ClAc-D-Tyr). Although the amino acid residue adjacent to X1 including X2, X3, X4 and X5 is not particularly limited if the prenylation enzyme of the present invention is used, it is preferably an amino acid residue promoting prenylation of the amino acid residue X1 to be prenylated from the standpoint of improving the reaction efficiency of prenylation.

X1 is W or a derivative thereof.

The following X2 to X5 and the following motifs are shown as examples when KgpF is used as a prenylation enzyme. When a prenylation enzyme other than KgpF is used, preferable X2 to X5 and the motif can be set by, as described above, comprehensively preparing peptides in a free-cell translation system and performing an efficiency confirmation test by in vitro prenylation with a predetermined prenylation enzyme.

X2 is preferably V, T, I, A, P, Q, N, S, F, H, W, L, or G, or a derivative of any of them, more preferably V, T, I, A, P, Q, or N or a derivative of any of them, still more preferably V, T, I, A, or N, or a derivative of any of them.

X3 is preferably G, W, S, A, L, F, T, R, N, Y, I, V, H, Q, K, D, or E, or a derivative of any of them, more preferably G, W, S, A, L, F, T, R, N, or Y, or a derivative of any of them, still more preferably G, W, or S, or a derivative of any of them.

X4 is preferably G, N, Y, I, R, E, F, S, W, H, D, T, P, A, V, L, Q, or K, or a derivative of any of them. X5 is preferably G, N, Y, I, R, E, F, S, W, H, D, T, P, A, V, L, Q, K, or C or a derivative of any of them.

For example, when KgpF is used as a prenylation enzyme, the prenylation enzyme can prenylate a peptide having the motif shown specifically below. In the following motifs, y means ClAc-D-Tyr.

VWG

VWW

VWS

VWI

VWC

VWT

VWR

VWQ

VWN

VWF

VWK

VWD

VWH

TWG

TWW

TWS

TWT

TWP

TWL

TWK

TWD

TWN

TWH

IWG

IWW

IWS

IWR

IWI

IWP

IWT

IWN

IWQ

IWE

IWH

AWG

AWW

AWS

AWH

AWV

AWD

AWL

AWT

AWR

AWQ

AWI

PWG

PWW

PWS

PWP

PWW

PWR

PWA

PWL

PWN

PWY

PWI

PWK

QWS

QWA

NWG

NWW

NWA

NWL

NWF

NWT

NWR

NWN

NWY

NWI

NWV

NWH

NWQ

NWK

NWD

NEW

NWP

NWS

SWS

FWF

FWS

HWS

HWG

HWD

WWQ

WWS

LWV

LWS

LWC

GWS

GWT

DWF

DWS

RWR

RWC

YWS

EWS

EWP

KWS

For example, when KgpF is used as a prenylation enzyme, the prenylation enzyme can also prenylate a peptide containing the motif shown below more specifically. In the following motif, y means ClAc-D-Tyr.

(SEQ ID NO: 57) GVWGG (SEQ ID NO: 58) GVWWG (SEQ ID NO: 59) GVWSG (SEQ ID NO: 60) EVWNL (SEQ ID NO: 61) RVWGP (SEQ ID NO: 62) RVWGC (SEQ ID NO: 63) RVWGY (SEQ ID NO: 64) HVWGD (SEQ ID NO: 65) DVWGY (SEQ ID NO: 66) DVWIF (SEQ ID NO: 67) YVWGR (SEQ ID NO: 68) YVWGH (SEQ ID NO: 69) HVWGT (SEQ ID NO: 70) TVWGY (SEQ ID NO: 71) YVWGP (SEQ ID NO: 72) PVWGC (SEQ ID NO: 73) HVWSL (SEQ ID NO: 74) AVWDH (SEQ ID NO: 75) SVWTC (SEQ ID NO: 76) AVWRS (SEQ ID NO: 77) yVWNV (SEQ ID NO: 78) AVWQT (SEQ ID NO: 79) RVWKL (SEQ ID NO: 80) TVWQQ (SEQ ID NO: 81) SVWSI (SEQ ID NO: 82) DVWQT (SEQ ID NO: 83) HVWHQ (SEQ ID NO: 84) EVWNL (SEQ ID NO: 85) GTWGG (SEQ ID NO: 86) GTWWG (SEQ ID NO: 87) GTWSG (SEQ ID NO: 88) HTWST (SEQ ID NO: 89) STWTS (SEQ ID NO: 90) NTWWQ (SEQ ID NO: 91) ITWPL (SEQ ID NO: 92) GTWLY (SEQ ID NO: 93) NTWKV (SEQ ID NO: 94) FTWDN (SEQ ID NO: 95) TTWDE (SEQ ID NO: 96) ETWNA (SEQ ID NO: 97) TTWNA (SEQ ID NO: 98) VTWNS (SEQ ID NO: 99) VTWNA (SEQ ID NO: 100) NTWNS (SEQ ID NO: 101) ETWHN (SEQ ID NO: 102) STWGS (SEQ ID NO: 103) LTWGA (SEQ ID NO: 104) STWGH (SEQ ID NO: 105) VTWKG (SEQ ID NO: 106) ATWNA (SEQ ID NO: 107) GIWGG (SEQ ID NO: 108) GIWWG (SEQ ID NO: 109) GIWSG (SEQ ID NO: 110) RIWRY (SEQ ID NO: 111) IIWII (SEQ ID NO: 112) GIWGS (SEQ ID NO: 113) QIWTH (SEQ ID NO: 114) RIWRK (SEQ ID NO: 115) KIWNS (SEQ ID NO: 116) TIWII (SEQ ID NO: 117) RIWQV (SEQ ID NO: 118) GIWEK (SEQ ID NO: 119) SIWSQ (SEQ ID NO: 120) HIWTD (SEQ ID NO: 121) IIWHR (SEQ ID NO: 122) QIWPN (SEQ ID NO: 123) GAWGG (SEQ ID NO: 124) GAWWG (SEQ ID NO: 125) GAWSG (SEQ ID NO: 126) SAWHG (SEQ ID NO: 127) PAWVR (SEQ ID NO: 128) YAWDQ (SEQ ID NO: 129) YAWLR (SEQ ID NO: 130) QAWTK (SEQ ID NO: 131) NAWTG (SEQ ID NO: 132) YAWRS (SEQ ID NO: 133) LAWQI (SEQ ID NO: 134) KAWIC (SEQ ID NO: 135) KAWHR (SEQ ID NO: 136) GPWGG (SEQ ID NO: 137) GPWWG (SEQ ID NO: 138) GPWSG (SEQ ID NO: 139) RPWVP (SEQ ID NO: 140) QPWPE (SEQ ID NO: 141) TPWVC (SEQ ID NO: 142) WPWRT (SEQ ID NO: 143) VPWAE (SEQ ID NO: 144) KPWLK (SEQ ID NO: 145) KPWNY (SEQ ID NO: 146) RPWYC (SEQ ID NO: 147) SPWID (SEQ ID NO: 148) DPWKS (SEQ ID NO: 149) NNWSP (SEQ ID NO: 150) NNWST (SEQ ID NO: 151) GNWSY (SEQ ID NO: 152) YNWSQ (SEQ ID NO: 153) INWSL (SEQ ID NO: 154) RNWSY (SEQ ID NO: 155) RNWSR (SEQ ID NO: 156) FNWSF (SEQ ID NO: 157) GNWSG (SEQ ID NO: 158) NNWST (SEQ ID NO: 159) GNWGG (SEQ ID NO: 160) GNWWG (SEQ ID NO: 161) GNWAG (SEQ ID NO: 162) GNWLG (SEQ ID NO: 163) GNWFG (SEQ ID NO: 164) GNWTG (SEQ ID NO: 165) GNWRG (SEQ ID NO: 166) GNWNG (SEQ ID NO: 167) GNWYG (SEQ ID NO: 168) GNWIG (SEQ ID NO: 169) GNWVG (SEQ ID NO: 170) GNWHG (SEQ ID NO: 171) GNWQG (SEQ ID NO: 172) GNWKG (SEQ ID NO: 173) GNWDG (SEQ ID NO: 174) GNWEG (SEQ ID NO: 175) NNWPT (SEQ ID NO: 176) PNWRF (SEQ ID NO: 177) FFWFF (SEQ ID NO: 178) GFWSG (SEQ ID NO: 179) GLWSG (SEQ ID NO: 180) YLWSK (SEQ ID NO: 181) yLWVL (SEQ ID NO: 182) NDWST (SEQ ID NO: 183) YDWFC (SEQ ID NO: 184) NEWST (SEQ ID NO: 185) HEWPW (SEQ ID NO: 186) NKWST (SEQ ID NO: 187) NYWST (SEQ ID NO: 188) GSWSG (SEQ ID NO: 189) ySWSP (SEQ ID NO: 190) ISWNE (SEQ ID NO: 191) GWWSG (SEQ ID NO: 192) TWWQR (SEQ ID NO: 193) GGWSG (SEQ ID NO: 194) TGWTR (SEQ ID NO: 195) GHWSG (SEQ ID NO: 196) FHWGL (SEQ ID NO: 197) IHWDC (SEQ ID NO: 198) GQWSG (SEQ ID NO: 199) YQWAC (SEQ ID NO: 200) YRWRV

For example, when OltF is used as a prenylation enzyme, the prenylation enzyme can also prenylate a peptide containing the motif shown below specifically. In the following motif, y means ClAc-D-Tyr.

NWS

IWR

PWV

VWI

SWN

NWR

yWY

AWD

VWN

RWG

FWF

GWG

YWY

NWN

VWV

FWL

LWV

DWF

Further, for example when OltF is used as a prenylation enzyme, the prenylation enzyme can also prenylate a peptide containing the motif shown below specifically.

(SEQ ID NO: 201) NNWSP (SEQ ID NO: 202) NNWST (SEQ ID NO: 203) RIWRY (SEQ ID NO: 204) RPWVP (SEQ ID NO: 205) DVWIF (SEQ ID NO: 206) ISWNE (SEQ ID NO: 207) PNWRF (SEQ ID NO: 208) YAWDQ (SEQ ID NO: 209) EVWNL (SEQ ID NO: 210) GRWGY (SEQ ID NO: 211) FFWFF (SEQ ID NO: 212) GGWGG (SEQ ID NO: 213) YYWYY (SEQ ID NO: 214) NNWNN (SEQ ID NO: 215) VVWVV (SEQ ID NO: 216) YFWLP (SEQ ID NO: 217) GLWVP (SEQ ID NO: 218) SDWFW (SEQ ID NO: 219) WFWLP

More specifically, at least peptides having the following amino acid sequence, as well as peptides shown later in Examples, can be prenylated.

Cyclic  (SEQ ID NO: 386) [WLNGDNNWSTP] Cyclic (SEQ ID NO: 382) [TSQIWGSPVP] Cyclic (SEQ ID NO: 383) [SAQWQNFGVP] (SEQ ID NO: 384) WLNGDNNWSTP (SEQ ID NO: 385) WLNGDNNWSTPAYDG

This can be understood from the finding that for example KgpF prenylates two Trp residues contained in a main-chain cyclized peptide (Cyclic [WLNGDNNWSTP]) formed from a precursor peptide (KgpE) of Kawaguchipeptin A and composed of 11 residues to form a natural product Kawaguchipeptin A (refer to Tetrahedron, 1996, 52, 9025. ACIE, 2016, 55, 3596. Org. Biomol. Chem., 2016, 14, 9639.). The prenylation enzyme can also prenylate Fmoc-Trp-OH though at low efficiency. In the above description, Cyclic[ ] and c[ ] means that it is an amino acid sequence of a cyclic peptide and a left-end amino acid residue and a right-end amino acid residue in the bracket bond to each other.

Although the concentration of the prenylation enzyme is not particularly limited because it differs depending on the expression and purification conditions of the enzyme, it is, for example, from 0.1 to 1000 μM. The concentration of the prenylation enzyme can be made smaller by adjusting the expression and purification conditions of the enzyme.

(Peptide Library Having Phenotype Displayed on Genotype)

A display method is a system capable of linking a phenotype to a genotype encoding the sequence thereof through non-covalent bonding or covalent bonding to display the phenotype on the genotype and performing enrichment and amplification (selection) of active species by using a replication system reconstructed in a test tube.

Examples of it include phage display with Escherichia coli as a replication medium and yeast display.

In vitro display without using a prokaryotic or eukaryotic organism as a medium can also be used and the in vitro display permits more diverse library search than phage display. Examples of the in vitro display include ribosome display, cDNA display, and mRNA display.

(Method of Producing Peptide-Genotype Library)

One aspect of the present invention is a method of producing a peptide-genotype complex library including a step of preparing a peptide-genotype library including genotype-peptide complexe(s) and a step of bringing the peptide-genotype library into contact with a prenylation enzyme and prenylating at least one Trp or derivative thereof.

(Method of Producing Peptide-Genotype Library)

As the display method of the present invention, any method can be used, and mRNA display can be used preferably.

Described specifically, the method of producing a peptide-genotype complex library is preferably a method of producing a peptide-mRNA complex library. The step of preparing a peptide-genotype library includes a step of preparing a peptide-mRNA library by the mRNA display method; and the step of preparing a peptide-mRNA library includes a step of binding puromycin to the 3′ end of each RNA of the mRNA library to produce a puromycin-bound mRNA library and a step of translating the puromycin-bound mRNA library in a cell-free translation system and preparing a peptide-mRNA library including peptides having an amino acid sequence containing at least one Trp or derivative thereof.

The step of producing a puromycin-bound mRNA library is performed, more specifically, by binding puromycin to the downstream region of ORF (Open reading frame) of each mRNA when a mRNA library is prepared in the method of producing a peptide library described above. Puromycin may be bound to mRNA via a linker composed of a peptide or nucleic acid. An mRNA-peptide complex is formed by binding puromycin to the downstream region of ORF of mRNA and incorporating puromycin in a ribosome which has translated ORF of mRNA. Such a peptide-mRNA complex allows association between genotype and phenotype so that it can be applied to in vitro display.

(Peptide Library)

The present invention embraces a peptide library produced by the above-described production method. More specifically, the peptide library of the present invention includes an aspect of a peptide library and an aspect of a peptide-mRNA complex library including the above-described cyclic peptides.

(Screening Method)

One aspect of the present invention is a screening method for identifying a peptide that binds to a target material, including a step of bringing a peptide library and/or peptide-genotype complex library, preferably a peptide-mRNA complex library, each produced by the above-described method, into contact with a target material and a step of selecting a peptide that binds to the target material. The contact step in the screening method of the present invention may be performed by bringing a peptide library and/or peptide-genotype complex library, preferably a peptide-mRNA complex library, each produced by the above-described method into contact with a target material, followed by incubation.

The target material is not particularly limited in the present specification and examples include low molecular weight compounds, high molecular weight compounds, nucleic acids, peptides, proteins, sugars, lipids and the like. In particular, the library of the present invention can also be applied to the case where a target material has protease activity or is an intracellular molecule.

The target material immobilized onto, for example, a solid-phase support may be brought into contact with the peptide library and/or peptide-mRNA complex library according to the present invention. The term “solid-phase support” as used herein is not particularly limited insofar as it can immobilize the target material onto itself. Examples include microtiter plates, substrates, and beads made of glass, a metal, a resin or the like, nitrocellulose membranes, nylon membranes, and PVDF membranes. The target material can become immobilized onto such a solid-phase support in a known manner.

The target material and the library are brought into contact with each other in a buffer selected as needed and they are interacted while controlling pH, temperature, time and the like.

The screening method of the present invention includes a step of selecting a peptide that has bound to the target material. The peptide that has bound to the target material can be selected, for example, by labeling peptides detectably by a known method and after the contact step, washing the surface of the solid-phase support with a buffer, and then detecting the compound that has bound to the target material.

Examples of a detectable label include enzymes such as peroxidase, alkaline phosphatase and the like, radioactive substances such as 25I, 131I, 35S, 3H and the like, fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethyl rhodamine isothiocyanate, near infrared fluorescent materials and the like, light-emitting substances such as luciferase, luciferin, aequorin and the like, and nanoparticles such as gold colloid, quantum dot and the like. When an enzyme is used as the label, detection can also be achieved by bringing the substrate of the enzyme into contact with the enzyme to cause color development. It is also possible to detect by binding biotin to the peptide and then binding avidin or streptavidin labeled with an enzyme or the like.

The screening method can not only detect or analyze the presence/absence or degree of binding but also analyze the enhanced or inhibited activity of the target material and thereby identify a peptide having such enhancing or inhibiting activity. Such a method also allows identification of a peptide having physiological activity and useful as a drug.

In the screening method of the present invention, a display method can be applied to the step of selecting a peptide that binds to a target material and usable examples of such a display method include phage display using Escherichia coli as a replication medium, yeast display, and ribosome display, cDNA display and mRNA display, which are each an in vitro display not using a prokaryotic or eukaryotic organism as a medium. Particularly when the peptide-mRNA complex library is used in the screening method of the present invention, the mRNA display method can be applied to the step of selecting a peptide that binds to the target material. The term “mRNA display method” as used herein means a method of, in the translation of mRNA, binding the mRNA to a synthesized peptide to allow association between a phenotype (peptide) and a nucleic acid sequence (mRNA).

In the display method, a peptide-genotype complex library is brought into contact with a target molecule and after incubation, a peptide-genotype complex group bound to the target molecule is selected. This step can be carried out, similar to that used for the above-described selection of a peptide bound to a target material, for example by fixing a target material onto the surface of a solid phase and selecting a peptide-genotype complex trapped on the surface of the solid phase.

When as the display method, the mRNA display method is used, a group of cDNAs can be obtained by subjecting a group of peptide-mRNA complexes to a reverse transcription reaction. This cDNAs encode cyclic peptides binding to a target molecule.

By amplifying the cDNA group and transcribing the resulting group, an mRNA library can be obtained again. In the mRNA library thus obtained again, the concentration of a molecule that binds to the target molecule becomes higher than that of the first mRNA library. The molecule that binds to the target molecule can therefore be enriched gradually by repeating the above step in a plurality of times.

The amino acid sequence of the enriched cyclic peptide can be determined by analyzing the sequence of the cDNA so that a cyclic peptide having high affinity for the target molecule can be produced based on the sequence information.

The RaPID system (Yamagishi, Y. et al., Chemistry & biology, 2011, 18 (12), 1562-70) is one example of a screening system having a FIT system, used in Examples described later, and mRNA display in combination. The RaPID system having a genetic code reprogramming technology using the FIT system and the mRNA display method in combination has enabled screening with a peptide library including nonproteins.

(Screening Kit)

The present invention also provides a peptide screening kit. One aspect of the screening kit of the present invention includes a peptide library and/or peptide-mRNA complex library produced by the production method of the present invention.

The screening kit of the present invention includes, in addition to the above-described peptide library and/or the peptide-mRNA complex library, a reagent and apparatus necessary for detecting binding of the peptide or peptide-mRNA complex and a target material. Examples of such a reagent and apparatus include, but not limited to, solid-phase supports, buffers, labeling reagents, enzymes, enzyme reaction terminator solutions, and microplate readers.

(Method of Producing Peptide)

The present invention also provides a method of producing a prenylated peptide, including a step of bringing a peptide having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating the at least one Trp residue or derivative residue thereof.

Although a method of obtaining the peptide having an amino acid sequence containing at least one Trp or derivative thereof is not particularly limited, examples include chemical synthesis, preferably a method of synthesizing using a solid-phase synthesis method. One of the preferred aspects of the method of producing a peptide according to the present invention includes a step of synthesizing, by chemical synthesis, a peptide having an amino acid sequence containing at least one Trp or derivative thereof and a step of bringing the peptide having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating the at least one Trp residue or derivative residue thereof.

The method of producing a peptide according to the present invention can be carried out based on the above-described method of producing a peptide library according to the present invention. Described specifically, in the prenylation step, it is preferred to bring a peptide having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme in the presence of a donor for prenyl group in the presence or absence of a cofactor.

Although the donor for donating a prenyl group is not particularly limited, examples include dimethylallyl diphosphate (DMAPP), isoprenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and phytyl diphosphate (PDP). These donors for donating a prenyl group may be used either singly or in combination of two or more.

Although the cofactor is not particularly limited, examples include magnesium ion, copper ion, iron ion, manganese ion, molybdenum ion, nickel ion, selenium ion, zinc ion and the like. They may be used either singly or in combination of two or more. Of these, magnesium ion is preferred. Whether the cofactor is used or not in the method of producing a peptide according to the present invention may be selected as needed, depending on the prenylation enzyme to be used.

(Translation System)

In the present invention, the cell-free translation system may contain an elongator tRNA and may further contain an initiator tRNA. As described above, the present inventors have developed the translation system in which N1N2N3 encodes an arbitrary amino acid by codon reassignment making use of flexizyme. In a natural translation system, tRNAs having an anticodon corresponding to each amino acid are present and the tRNAs have intrinsic sequences respectively also in a region other than the anticodon loop.

When an arbitrary amino acid is reassigned to any of N1N2N3s by making use of flexizyme, all the tRNAs may be artificial. In this case, elongator tRNAs corresponding to respective N1N2N3s to be added to the translation system may have base sequences 80% or more, 85% or more, 88% or more, or 90% or more in full length identical to one another. This means that a group of elongator tRNAs almost equal in their sequences except for the sequences of anticodons may be used. The elongator tRNA group may have base sequences identical to one another except for anticodon loops. The elongator tRNAs corresponding to respective N1N2N3s to be added to the translation system may have sequences 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 98% or more, or 99% or more identical to one another except for anticodon loops.

The term “anticodon loop” as used herein means a loop portion of a single strand of a tRNA containing an anticodon. The sequence of the anticodon loop can be determined as needed by those skilled in the art so as to complement the codon-anticodon interaction.

The translation system thus obtained includes, as tRNAs, only one kind of initiator tRNAs and one kind of elongator tRNAs (that is, tRNAs having almost the same base sequence except for anticodons) so that the reactivity of the tRNAs becomes uniform and predetermined peptides can therefore be obtained with good reproducibility.

According to the translation system of the present invention, a library including 1×10¹² to 1×10¹³ or more respectively different peptides can be produced based on the diversity of N1N2N3 in the mRNA.

The translation system of the present invention may be used for transcription after addition of a factor necessary for transcription.

The translation system of the present invention is suited for use in the method of producing a peptide library according to the present invention described above.

All the disclosures of the patent documents and reference documents cited herein are incorporated herein in their entirety by reference.

EXAMPLES

The present invention will hereinafter be described specifically based on Examples, but the present invention is not limited to or by them. Those skilled in the art can change the present invention into various aspects without departing from the gist of the present invention and such a change is also embraced within the scope of the present invention.

1. FIT System v7S/v8.0.3

The FIT system has the following composition:

50 mM HEPES-KOH (pH 7.6); 12 mM magnesium acetate; 100 mM potassium acetate; 2 mM sperm idine; 20 mM creatinine phosphate; 2 mM DTT; 2 mM ATP; 2 mM GTP; 1 mM CTP; 1 mM UTP; 0.1 mM 10-formyl-5,6,7,8-tetrahydrofolic acid; 0.5 mM 15 proteinogenic amino acids other than Met, Lys, Gln, Trp, and Glu; 1.5 mg/ml E. coli total tRNA; 0.73 μM AlaRS; 0.03 μM ArgRS; 0.38 μM AsnRS; 0.13 μM AspRS; 0.02 μM CysRS; 0.06 μM GlnRS; 0.23 μM GluRS; 0.02 μM GlyRS; 0.02 μM HisRS; 0.4 μM IleRS; 0.04 LeuRS; 0.11 μM LysRS; 0.03 μM MetRS; 0.68 μM PheRS; 0.16 μM ProRS; 0.04 μM SerRS; 0.09 μM ThrRS; 0.03 μM TrpRS; 0.02 μM TyrRS; 0.02 μM VaIRS; 0.6 μM MTF; 2.7 μM IF1; 0.4 μM IF2; 1.5 μM IF3; 0.26 μM EF-G; 10 μM EF-Tu; 10 μM EF-Ts; 0.25 μM RF2; 0.17 μM RF3; 0.5 μM RRF; 0.1 μM T7 RNA polymerase; 4 pg/ml creatine kinase; 3 pg/ml myokinase; 0.1 μM pyrophosphatase; 0.1 μM nucleotidediphosphatase kinase, and 1.2 μM ribosome.

2. Preparation of ClAc-D/L-Trp/Tyr-tRNA^(fMet) _(CUA)

In 83.3 mM HEPES-KOH (pH 8.0), 3 μL of 83.3 μM tRNA^(fmet) _(CUA) and 3 μL of 83.3 μM flexizyme were heated at 95° C. for 2 minutes and then cooled to room temperature in 5 minutes. To the resulting RNA solution was added 2 μL of 3 M MgCl₂ and the resulting mixture was incubated on ice for 5 minutes. Then, 2 μL of 25 mM ClAc-D-Trp-CME, ClAc-L-Trp-CME, ClAc-D-Tyr-CME, or ClAc-L-Tyr-CME (in DMSO) was added to the reaction mixture, followed by incubation on ice for 2 hours. The reaction was terminated by adding 40 μL of 0.3 M sodium acetate (pH 5.2) and tRNA was collected by ethanol precipitation. Precipitate was washed twice with 70% ethanol and 0.1 M sodium acetate (pH 5.2) and once with 70% ethanol. After air drying for 10 minutes, the precipitate was dissolved in 1.0 μL of 1.0 mM sodium acetate and the resulting solution was used for the following translation reaction.

3. Prenylation Enzyme

With regards to each of KgpF and OltF, heterogeneous co-expression of a gene synthesized after optimizing the codon of a gene derived from a producing strain was performed in Escherichia coli, followed by purification to obtain a prenylation enzyme.

4. MALDI-TOF Analysis

The analysis was performed using UltrafleXtreme (Bruker Daltonics) or Autoflex II (Bruker Daltonics) and peptide calibration standard II (Bruker Daltonics).

In addition, reagents and the like disclosed in WO2014/181888 and WO2015/030014, particularly those disclosed in Examples thereof were used as needed also in the present Examples.

Example 1: Modification Test of Thioether-Closed Artificial Cyclic Peptide Having Sequence Element of Kawaguchipeptin, a Natural Substrate

Three kinds of thioether-closed artificial cyclic amino acid sequences YG280 to 282 derived from the sequence of Kawaguchipeptin, a natural substrate, (which may also be called “natural substrate sequence”) were designed.

(In the formula, shown in the parentheses is an amino acid sequence).

For three kinds of thioether-closed artificial cyclic peptides (YG280 to 282), peptides whose N-terminal Trp as an initiator residue had a steric configuration of D-form and those of L-form were synthesized. In short, six kinds in total of peptides were obtained by translation and synthesis under (Conditions 1) of the artificial translation system developed by the present inventors. The peptides thus obtained were then reacted with 14.7 μM KgpF at 37° C. for 18 hours in the system shown in (Conditions 2). “DMAPP” used in (Conditions 2) is dimethylallyl pyrophosphoric acid.

(Conditions 1)

-   -   FIT system: v7s/v8.0.3     -   19aa-Met: 0.5 mM     -   ClAc-D or L-Trp-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 pL scale

(Conditions 2)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 1))     -   KgpF: 14.7 μM     -   DMAPP: 2 mM     -   Total amount: 15.7 μL     -   Reaction: 37° C., 18 hours

The above reaction products were analyzed by MALDI-TOF-MS. The results were as shown below.

For all the three kinds of peptides tested, products having a molecular weight increased by that corresponding to prenylation were observed when they were treated with KgpF. This has suggested that the above peptides become a substrate of KgpF.

Although the peptides had a Trp at two positions therein, prenylation occurred at only one position.

Among the three kinds of peptides tested, the peptide having the YG280 sequence with a steric configuration of the N terminal Trp of D-form showed the most excellent modification efficiency by prenylation, so that the tests were continued further while focusing on the YG280 sequence.

Example 2: Modification of Thioether-Closed Artificial Cyclic Peptide with KgpF—Optimization of Enzyme Concentration

Based on the results of Example 1, the prenylation reaction conditions were optimized by using the peptide having a YG280 sequence and gradually increasing the concentration of KgpF.

The YG280 sequences having the steric configuration of the N terminal Trp serving as an initiation residue of D-form and those of L-form were synthesized, so that two kinds of peptides were prepared in total. These two kinds of artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 3) developed by the present inventors. The peptides thus obtained were then reacted with 53, 160, and 580 μM KgpF in a system shown in (Conditions 4) at 37° C. for 18 hours.

(Conditions 3)

-   -   FIT system: v7s/v8.0.3     -   19aa-Met: 0.5 mM     -   ClAc-D or L-Trp-tRNA^(fMet) _(CUA): 120 μM     -   Reaction: 37° C., 3 hours     -   Scale: 2.5 μL scale

(Conditions 4)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 3))     -   KgpF: 53, 160, 580 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

In the system using 160 μM KgpF, slight precipitation was found, while in the system using 580 μM KgpF, much precipitation was found.

The above reaction products were analyzed by MALDI-TOF-MS. The results are as shown below.

With an increase in the concentration of KgpF, a peak showing the modification product became more intense correspondingly.

The peptides having an N-terminal Trp in D-form as the steric configuration were superior in prenylation modification efficiency to those having that in L-form.

When the peptides having an N-terminal Trp in D-form as the steric configuration were reacted with 580 μM KgpF, almost all the peptides underwent prenylation modification (there were only few peaks showing non-prenylated peptides).

The peptides had a Trp at two positions but prenylation occurred only at one position.

It has been found that using a highly-concentrated KgpF enables efficient prenylation of a thioether-closed artificial cyclic peptide.

Example 3: Modification of Thioether-Closed Artificial Cyclic Peptide with KgpF—Identification of Modification Site

In Examples 1 and 2, prenylation occurred only at one position, though the peptide had a Trp at 2 positions. To determine the position where prenylation occurred, the peptides (YG298, 280) whose Trp residues in the YG280 sequence had each been mutated into Tyr and the peptide (YG290) having no Trp residue in a random region were designed and presence or absence of progression of modification with KgpF was assessed.

The above three kinds of artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 5) developed by the present inventors. The peptides thus obtained were then reacted with KgpF in the system shown below in (Conditions 6) at 37° C. for 18 hours.

(Conditions 5)

-   -   FIT system: v7s/v8.0.3     -   19aa-Met: 0.5 mM     -   ClAc-D or L-Trp-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

(Conditions 6)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 5))     -   KgpF: 160 or 580 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

The above reaction products were analyzed by MALDI-TOF-MS. As a result, it has been confirmed that no modified product was found even after the peptide YG298 having the N-terminal Trp residue remained therein or the peptide YG290-CM11 having no Trp residue in the peptide chain thereof was reacted with 580 μM KgpF, while the peptide ClAc-D-Tyr-initiated YG280 having the Trp residue remained in the chain (8th residue) was modified at an efficiency equal to that of the peptide of the original sequence (YG280). This has suggested that the Trp residue in the peptide chain of the peptide YG280 underwent prenylation modification.

Example 4: Research of Substrate Tolerance of KgpF—Modification of Peptides with Various Ring Sizes

The YG280 sequence derived from a natural substrate sequence was a thioether-closed cyclic peptide composed of 12 residues. It was therefore studied whether the thioether-closed cyclic peptides (YG310 and 311) having a shorter sequence and the thioether-closed cyclic peptides (YG312 to 314) having a longer sequence than that of YG280-1 used as a standard could be modified with KgpF or not.

The above six kinds of artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 7) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 8) at 37° C. for 18 hours.

(Conditions 7)

-   -   FIT system: v7s/v9.1     -   19aa-Met: 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

(Conditions 8)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 7))     -   KgpF: 160 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

The above reaction products were analyzed by MALDI-TOF-MS. As a result, it has been found that although there was an increase or decrease in the modification efficiency, depending on the ring size, all the sequences tested were modified with KgpF. This has proven that KgpF can modify thioether-closed artificial cyclic peptides having various ring sizes.

Example 5: Research of Substrate Tolerance of KgpF—Verification of Flanking Sequence of Trp (1)

In the natural substrate sequence and YG280, amino acid residues present upstream and downstream of Trp to be modified were Asn and Ser, respectively. The three kinds of amino acid sequences (YG300, 304, and 305) were designed by changing the flanking sequence of Trp and whether they could be modified with KgpF or not was studied.

The above three kinds of artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 9) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 10) at 37° C. for 18 hours.

(Conditions 9)

-   -   FIT system: v7s/v9.1     -   19aa-Met: 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

(Conditions 10)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 9))     -   KgpF: 160 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

The reaction products were analyzed by MALDI-TOF-MS. As a result, it was found that the three kinds of the artificial peptides tested were each modified by prenylation.

In addition, the following cyclic peptides (YG288-S2iL8, YG289-S2iD7, YG292-PaktiL5, and YG297-aMD4) were also obtained similarly by translation and synthesis and reacted with DMAPP in the presence of KgpF. It was observed by MALDI-TOF-MS that they were modified with a prenyl group.

Example 6: Research of Substrate Tolerance of KgpF—Verification of Flanking Sequence of Trp (2)

To closely investigate the influence of the flanking sequence of Trp on the modification efficiency with KgpF, 13 kinds of sequences having, instead of the flanking sequence of W, the NWS or NNWST sequence found in Kawaguchipeptin, a natural substrate, were designed (Table 5) and whether they could be modified with KgpF or not was studied.

TABLE 5 DMAPP adduct MALDI SEQ ID N template 0 1 2 mode NO YG300-CM11(RFG > NWS)

YCDVSGNWSYPC 2 4 0 LN 239 YG317-aMD5-NWS

YWYYNWSQTYKAFPC 1 4 0 LP 240 YG318-aMD5-NNWST

YWYNNWSTTYKAFPC 1 4 0 LP 241 YG319-PaktiL2-NWS

YWILINWSLVRRKC 1 4 0 LP 242 YG321-301-NWS

YRRNWSYKDDDDKC 1 4 0 LP 243 YG322-301-NNWST

YRNNWSTKDDDDKC 1 4 0 LP 244 YG323-302-NWS

YLNGDRNWSRPC 1 4 0 LP 245 YG280

YLNGDNNWSTPC 2 4 0 LP 220 YG304-YG280(FFWFF)

YLNGDFFWFFPC 4 3 0 LN 246 YG325-304-NWS

YLNGDFNWSFPC 3 3 0 LP 247 YG326-306-NWS

YLNGDGNWSCGC 4 3 0 LP 248 0: N/A, 1: Nothing, 2: Very weak intensity, 3: Medium intensity, 4: Strong intensity

The above artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 11) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 12) at 37° C. for 18 hours.

(Conditions 11)

-   -   FIT system: v7s/v9.1     -   19aa-Met: 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

(Conditions 12)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 1))     -   KgpF: 160 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

The above reaction products were analyzed by MALDI-TOF-MS. As a result, it has been found that a modification efficiency with KgpF showed a marked improvement by replacing the flanking sequence of W with the NWS or NNWST sequence found in the natural substrate.

Example 7: Research of Substrate Tolerance of KgpF—Verification of Flanking Sequence of Trp (3)

What kind of a flanking sequence of W would make the resulting peptide excellent and preferable in prenylation with KgpF was studied further comprehensively.

Here, the following mutants were prepared comprehensively by replacing the flanking sequence of W of peptide YG326 with another amino acid residue and whether they could be prenylated with KgpF or not was studied.

The peptides in which N (X in Mutant I of YG326) upstream of W contained in the sequence of peptide YG326 and S (X in Mutant II of YG326) downstream of the W were replaced with various amino acid residues shown along the abscissa of the graphs shown in FIG. 1 and FIG. 2 were synthesized as in Example 6 and a prenylation reaction was performed as in Example 6. The reaction products were analyzed by MALDI-TOF-MS. The results are as shown below.

The graphs shown in FIG. 1 and FIG. 2 are histograms including an apparent modification efficiency on the mass spectrum which was calculated by quantitatively determining the respective peak intensities of an unmodified peptide and a modified peptide in mass spectrum of each reaction product. In the graph of FIG. 1, with regards to the amino acids upstream of W, amino acid residues are aligned in the descending order of modification efficiency given by them. In the graph of FIG. 2, with regards to the amino acids downstream of W, amino acid residues are aligned in the descending order of modification efficiency given by them.

The level of the modification efficiency of N which is a natural upstream sequence is indicated by a dotted line.

The following respective compounds having D, E, K and Y as the amino acid upstream of W and the following compound having P as the amino acid downstream of W were also synthesized as in Example 6 and a prenylation reaction was performed as in Example 6.

Observation by MALDI-TOF-MS showed that the above compounds YG280-DWS, YG280-EWS, YG280-KWS, YG280-YWS, and YG280-NWP were modified with a propyl group.

It has been found in general that amino acid residues having a variety of properties such as acidity, basicity, affinity, hydrophobicity and the like were tolerated both at the upstream position and the downstream position.

Example 8: Research of Substrate Tolerance of KgpF—Verification of Flanking Sequence of Trp (4)

Based on the results obtained in Example 7, it becomes possible to design some flanking sequences giving a higher modification efficiency than the natural NWS flanking sequence. Here, the 10 kinds of flanking sequences were newly designed (the abscissa of FIG. 3), which were obtained by multiplying, as an upstream sequence, the 5 kinds of amino acids superior to natural N in modification efficiency and, as a downstream sequence, the 2 kinds of amino acids superior to natural S in modification efficiency. The peptides were each synthesized as in Example 6 and a prenylation reaction was performed as in Example 6.

The above reaction products thus obtained were analyzed by MALDI-TOF-MS. As a result, it has been found that almost all the peptides thus analyzed showed a modification efficiency higher than that of the peptide having a natural NWS sequence (FIG. 3).

Example 9: Research of Substrate Tolerance of KgpF—Verification of Flanking Sequence of Trp (5)

Based on the results of the modification efficiency of the flanking sequence obtained in Example 7, a peptide whose amino acid residue upstream of W was fixed to the most preferable Val and amino acid sequence on the downstream side was changed was designed. Designed were mutants obtained by replacing the X portion of VWX with the 6 kinds of amino acids (G, W, S, F, N, and K). The resulting peptides were synthesized as in Example 6 and a prenylation reaction was performed as in Example 6.

The above reaction products thus obtained were analyzed by MALDI-TOF-MS. As a result, it has been found that they showed a modification efficiency equal or greater than the natural NWS (FIG. 4). All the Examples described above have suggested that when the amino acid upstream of Trp is a modifiable amino acid (V, T, I, A or P), the Trp is prenylated by KgpF without depending on the downstream amino acid residue. It has been verified as a result of comprehensive substrate tolerance research that KgpF is a Trp prenylation enzyme having very high substrate tolerance.

Example 10: KgpF Modification Reaction in Amino Acid Sequence Containing a Plurality of Trps

The artificial cyclic peptides Clone-M1 to M9 shown below were obtained by translation and synthesis in the artificial translation system (Conditions 13) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 14) at 0° C. for 20 hours.

(In the above formulas, “y” represents D-Tyr).

(Conditions 13)

-   -   FIT system: v8s/v11.0     -   19aa-Met: 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

After the above reaction was performed, the reaction product was incubated at 25° C. for 12 minutes.

Then, a cyclization reaction was performed under the following conditions.

-   -   Translated mixture amount: 2.5 μL (mixture obtained by the above         reaction)     -   EDTA: 18 mM     -   Reaction: 42° C., 60 min.     -   Scale: 2.75 μL scale

(Conditions 14)

-   -   Translated mixture amount: 2.75 μL (mixture obtained under         (Conditions 13))     -   KOH: 6.0 mM     -   Mg(OAc)₂: 9.0 mM     -   Tris-HCl (pH 8.3) 15 mM     -   KgpF: 160 μM     -   DMAPP: 2 mM     -   Reaction: 0° C., 20 hours

Shown in Table 6 are 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.

TABLE 6 Number of Number of Number of Number of additions 0 additions 1 additions 2 additions 3 Clone-M1 1 1 5 N/A Clone-M2 1 5 1 N/A Clone-M3 1 1 4 N/A Clone-M4 1 4 1 1 Clone-M5 3 5 1 N/A Clone-M6 1 5 5 N/A Clone-M7 1 4 5 N/A Clone-M8 1 1 5 1 Clone-M9 1 1 5 N/A

Example 11: Improvement in Modification Reaction Efficiency by Changing KgpF

Reaction of the artificial cyclic peptide shown in Table 7 was made under conditions similar to (Conditions 11) and (Conditions 12) except that v8s/v11.1 was used as FIT system, KgpF v 3.1 was used as a prenylation modification enzyme, and the concentration of the KgpF v 3.1 was set at 10 μM. Here, KgpF v 3.1 is an enzyme having a higher purification degree and more improved specific activity than KgpF used under (Conditions 12).

Shown in Table 7 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.

TABLE 7 The number of prenyl KgpF v 3.1 groups added Sequence template (μM) 0 1 2 3 SEQ ID YG281 YLNGDNNWSTPC 10 1 5 N/A N/A 265 YG282 YLNGDNNWSPC 10 1 5 N/A N/A 266 YG288-S2iL8 YSNFRIWRYSNSSC 10 1 5 N/A N/A 267 YG289-S2iD7 YHDYRIWRYHTYPC 10 1 5 N/A N/A 268 YG291-PaktiL2 YWILITWPLVRRKC 10 4 4 N/A N/A 269 YG292-PaktiL5 YIRRPWVPIIYLGC 10 1 5 N/A N/A 270 YG293-MaL4 YTFRDVWIFYGSLLSRC 10 1 5 N/A N/A 271 YG295-aML5 YISWNEFNSPNWRFITC 10 1 5 1 N/A 272 YG296-aMD5 YWYYAWDQTYKAFPC 10 5 3 1 N/A 273 YG297-aMD4 YRQFNRRTHEVWNLDC 10 1 5 N/A N/A 274

Example 12: Modification of Artificial Cyclic Peptides Containing VW Sequence and Having Various Ring Sizes

The artificial cyclic peptides shown in Table 8 and Table 9 were obtained by translation and synthesis in the artificial translation system (Conditions 15) developed by the present inventors. The peptides thus obtained were then reacted with 60 μM KgpF in the system shown in (Conditions 16) at 37° C. for 18 hours.

(Conditions 15)

-   -   FIT system: v8s/v11.1     -   19aa-Met: 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

(Conditions 16)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 15))     -   KgpF: 60 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

Shown in Table 8 and Table 9 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.

TABLE 8  The number of prenyl KgpF groups added SEQ Sequence Initiator template (μM) 0 1 2 3 ID YG386 ClAc-(D)Tyr VWCYPRG 60 1 5 N/A N/A 275 YG387 ClAc-(D)Tyr VW

CYPR 60 1 5 N/A N/A 276 YG388 ClAc-(D)Tyr RVW

CYP 60 1 5 N/A N/A 277 YG389 ClAc-(D)Tyr RVW

PCY 60 1 5 N/A N/A 278 YG390 ClAc-(D)Tyr RVW

YPC 60 1 5 N/A N/A 279

TABLE 9 The number of KgpF prenyl groups added SEQ Sequence Initiator template (μM) 0 1 2 3 ID YG391 ClAc-(D)Tyr YRV

YPC 60 1 5 N/A N/A 280 YG392 ClAc-(D)Tyr YRV

YHPC 60 1 5 N/A N/A 281 YG393 ClAc-(D)Tyr DYRV

YHPC 60 1 5 N/A N/A 282 YG394 ClAc-(D)Tyr DYRV

YHTPC 60 1 5 N/A N/A 283 YG395 ClAc-(D)Tyr HDYRV

YHTPC 60 1 5 N/A N/A 284 YG396 ClAc-(D)Tyr HDYRV

YHTYPC 60 1 5 N/A N/A 285 YG397 ClAc-(D)Tyr HNDYRV

YHTYPC 60 1 5 N/A N/A 286 YG398 ClAc-(D)Tyr HNDYRV

YHTYQPC 60 1 5 N/A N/A 287 YG399 ClAc-(D)Tyr HNQDYRV

YHTYQPC 60 1 5 N/A N/A 288 YG400 ClAc-(D)Tyr HNQDYRV

YHTYQNPC 60 1 5 N/A N/A 289 YG401 ClAc-(D)Tyr HNQNDYRV

YHTYQNPC 60 1 5 N/A N/A 290

Example 13: Modification of Artificial Cyclic Peptides Containing VW or WG Sequence and Having W at Various Positions

The artificial cyclic peptides shown in Table 10 were obtained by translation and synthesis in the artificial translation system (Conditions 17) developed by the present inventors. The peptides thus obtained were then reacted with 60 μM KgpF in the system shown in (Conditions 18) at 37° C. for 18 hours.

(Conditions 17)

-   -   FIT system: v8s/v11.1     -   19aa-Met: 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

(Conditions 18)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 17))     -   KgpF: 60 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

Shown in Table 10 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.

TABLE 10 The number of KgpF prenyl groups added SEQ Sequence Initiator template (μM) 0 1 2 3 ID YG402 ClAc-(D)Tyr

HDYRVYHTYPC 60 5 1 N/A N/A 291 YG403 ClAc-(D)Tyr V

HDYRYHTYPC 60 1 5 N/A N/A 292 YG404 ClAc-(D)Tyr HV

DYRYHTYPC 60 2 5 N/A N/A 293 YG405 ClAc-(D)Tyr HDV

YRYHTYPC 60 1 5 N/A N/A 294 YG406 ClAc-(D)Tyr HDYV

RYHTYPC 60 1 5 N/A N/A 295 YG407 ClAc-(D)Tyr HDYRYV

HTYPC 60 1 5 N/A N/A 296 YG408 ClAc-(D)Tyr HDYRYHV

TYPC 60 1 5 N/A N/A 297 YG409 ClAc-(D)Tyr HDYRYHTV

YPC 60 1 5 N/A N/A 298 YG410 ClAc-(D)Tyr HDYRYHTYV

PC 60 1 5 N/A N/A 299 YG411 ClAc-(D)Tyr HDYRYHTYPV

C 60 1 5 N/A N/A 300 YG412 ClAc-(D)Tyr HDYR

YHTYPV

C 60 1 5 N/A N/A 301

Example 14: Modification of Artificial Cyclic Peptides Having Various Peptide Sequences

The artificial cyclic peptides shown below were obtained by translation and synthesis in the artificial translation system (Conditions 19) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 20) at 0° C. for 20 hours.

The reaction products were analyzed by MALDI-TOF-MS. As a result, it has been confirmed that the artificial cyclic peptides shown below were modified by prenylation. As described above, the fact that prenylation proceeded in peptides having various sequences means that a peptide library including peptides having higher cell membrane permeability can be obtained efficiently.

(Conditions 19)

-   -   FIT system: v8s/11.0     -   19aa-Met: 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

After the above reaction was performed, the reaction product was incubated at 25° C. for 12 minutes.

Then, a cyclization reaction was performed under the following conditions.

-   -   Translated mixture amount: 2.5 μL (mixture obtained by the above         reaction)     -   EDTA: 18 mM     -   Reaction: 42° C., 60 min.     -   Scale: 2.75 μL scale

(Conditions 20)

-   -   Translated mixture amount: 2.75 μL (mixture obtained under         (Conditions 19))     -   KOH: 6.0 mM     -   Mg(OAc)₂: 9.0 mM     -   Tris-HCl (pH 8.3): 15 mM     -   KgpF: 160 μM     -   DMAPP: 2 mM     -   Reaction: 0° C., 20 hours

(In the above formulas, “y” represents D-Tyr).

(In the above formulas, “y” represents D-Tyr).

(In the above formulas, “y” represents D-Tyr).

(In the above formulas, “y” represents D-Tyr).

(In the above formulas, “y” represents D-Tyr).

Example 15: Modification Test of Thioether-Closed Artificial Cyclic Peptide with OltF

In order to prove that not only KgpF but also another enzyme having homology with it could modify a thioether-closed artificial cyclic peptide, it was studied whether or not a thioether-closed artificial cyclic peptide could be modified with an OltF enzyme.

The 6 kinds of thioether-closed artificial cyclic amino acid sequences (YG413 to 417 and 419) derived from the sequence of OscE1 and OscE2 presumed to be natural substrates of OltF were designed.

The above artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 21) developed by the present inventors. The peptides thus obtained were then reacted with 24 or 72 μM OltF in the system shown in (Conditions 22) at 37° C. for 18 hours.

(Conditions 21)

-   -   FIT system: v7s/v9.1     -   19aa-Met: 0.5 mM     -   ClAc-D or L-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

(Conditions 22)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 21))     -   OltF: 24, 72 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

The above reaction products thus obtained were analyzed by MALDI-TOF-MS. As a result, a product modified by prenylation was observed in any of the peptides. It has been found from the above fact that OltF, similar to KgpF, has an ability of modifying not only a natural main-chain cyclized peptide but also a thioether-closed artificial cyclic peptide. This has suggested that a group of enzymes having high homology with OltF or KgpF can modify a thioether-closed artificial cyclic peptide similarly to them and therefore can be used as the prenylation enzyme of the present invention.

Example 16: Modification Test of Thioether-Closed Artificial Cyclic Peptide with OltF

The artificial cyclic peptides shown in Table 11 and Table 12 were obtained by translation and synthesis in the artificial translation system (Conditions 23) developed by the present inventors. The peptides thus obtained were then reacted with 9.13 μM OltF in the system shown in (Conditions 24) at 37° C. for 18 hours. It is to be noted that OltF v 2.1 is an enzyme prepared from OltF used in (Conditions 22) while changing a purification manner.

(Conditions 23)

-   -   FIT system: v8s/v11.1     -   19aa-Met: 0.5 mM     -   ClAc-D or L-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

(Conditions 24)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 23))     -   OltF v 2.1: 9.13 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 18 hours

Shown in Table 11 and Table 12 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.

TABLE 11 The number of prenyl OltF v groups added SEQ Sequence Initiator template 2.1 0 1 2 3 ID YG281 ClAc-(D)Tyr YLNGDNNWSPC 9.13 μM 5 3 N/A N/A 336 YG282 ClAc-(D)Tyr YLNGDNNWSTC 9.13 μM 1 4 N/A N/A 337 YG288 ClAc-(D)Tyr YSNFRIWRYSNSSC 9.13 μM 1 5 N/A N/A 338 YG289 ClAc-(D)Tyr YHDYRIWRYHTYPC 9.13 μM 1 5 N/A N/A 339 YG292 ClAc-(D)Tyr YIRRPWVPIIYLGC 9.13 μM 2 5 N/A N/A 340 YG293 ClAc-(D)Tyr YTFRDVWIFYGSLLSRC 9.13 μM 3 4 N/A N/A 341 YG295 ClAc-(D)Tyr YISWNEFNSPNWRFITC 9.13 μM 4 3 1 N/A 342 YG296 ClAc-(D)Tyr YWYYAWDQTYKAFPC 9.13 μM 3 2 1 N/A 343 YG297 ClAc-(D)Tyr YRQFNRRTHEVWNLDC 9.13 μM 5 4 N/A N/A 344 YG299 ClAc-(D)Tyr YCDVSGRWGYFPC 9.13 μM 3 5 N/A N/A 345

Sequence The number of prenyl groups added

TABLE 12 The number of prenyl OLtF v groups added SEQ Sequence Initiator template 2.1 0 1 2 3 ID DNT061 ClAc-(D)Tyr YNNRERGSDFFWFFPC 9.13 μM 2 5 N/A N/A 346 DNT062 ClAc-(D)Tyr YNNRERGSDGGWGGPC 9.13 μM 3 5 N/A N/A 347 DNT063 ClAc-(D)Tyr YNNRERGSDYYWYYPC 9.13 μM 2 5 N/A N/A 348 DNT065 ClAc-(D)Tyr YNNRERGSDNNWNNPC 9.13 μM 3 5 N/A N/A 349 DNT066 ClAc-(D)Tyr YNNRERGSDVVWVVPC 9.13 μM 4 5 N/A N/A 350 DNT070 ClAc-(D)Tyr YISWNEFNSYFWLPITC 9.13 μM 1 5 1 N/A 351 DNT034 ClAc-(D)Tyr YNNRERGSDYFWLPPC 9.13 μM 1 5 N/A N/A 352 DNT057 ClAc-(D)Tyr YRQFNRRTHYFWLPDC 9.13 μM 1 5 N/A N/A 353

Example 17: Preparation of mRNA Library

(Design of mRNA Library)

As shown in Table 13, an mRNA library was designed.

TABLE 13 ... AUG XXX UGG NNK NNK NNK NNK NNK NNK NNK NNK NNK NNK UGU ... {close oversize brace} Group1 ... AUG NNK XXX UGG NNK NNK NNK NNK NNK NNK NNK NNK NNK UGU ... ... AUG NNK NNK XXX UOG NNK NNK NNK NNK NNK NNK NNK NNK UGU ... ... AUG NNK NNK NNK XXX UGG NNK NNK NNK NNK NNK NNK NNK UGU ... ... AUG NNK NNK NNK NNK XXX UGG NNK NNK NNK NNK NNK NNK UGU ... ... AUG NNK NNK NNK NNK NNK XXX UGG NNK NNK NNK NNK NNK UGQ ... {close oversize brace} Group2 ... AUG NNK NNK NNK NNK NNK NNK XXX UGG NNK NNK NNK NNK UGU ... ... AUG NNK NNK NNK NNK NNK NNK NNK XXX UGG NNK NNK NNK UGU ... ... AUG NNK NNK NNK NNK INK NNK NNK NNK XXX UGG NNK NNK UGU ... {close oversize brace} Group1 ... AUG NNK NNK NNK NNK NNK NNK NNK NNK NNK XXX UGO NNK UGU ... In Table 13, from top to bottom the sequence identifiers are SEQ ID NOs: 503 to 512.

In the mRNAs in Table 13, amino acids were reassigned based on the genetic code table shown below in Table 14. Here, Trp is reassigned to UGG of the mRNA in Table 13. XXX was RYU and CMG (RYU and CMG were present at 2:1 (RYU: CMG)) or RYU. It is to be noted that in RYU, Rs each independently represent G or A and Ys each independently represent U or C; in CMG, Ms each independently represent A or C; and in NNK, Ks each independently represent G or U, and N is an arbitrary base.

TABLE 14 U C A G U Phe Ser Tyr Cys U Phe Ser Tyr Cys C Leu Ser STOP STOP A Leu Ser STOP Trp G C Leu Pro His Arg U Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G A Ile Thr Asn Ser U Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G G Val Ala Asp Gly U Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G

(Preparation of mRNA Library)

<Composition of 1 mL Scale System>

-   -   MgCl₂: 2.5 mM     -   dNTPs: 0.25 mM     -   KCl: 50 mM     -   Tris-HCl (pH 9.0): 10 mM     -   Triton X-100: 1 μL     -   Primers: 250 nM (a primer containing T7YGM.F46 v1tr.F46 and the         sequence of the above library) Taq polymerase

<Extension of 1 mL Scale System>

Extension was achieved as shown in Table 15 by keeping at 95° C. for 1 minute, keeping at 61° C. for 1 minute and then keeping at 72° C. for 1 minute. Keeping at 61° C. for 1 minute and then keeping at 72° C. for 1 minute were performed with 5 cycles.

It is to be noted that the sequence of the primer T7YGM.F46 v1tr.F46 is as follows:

T7YGM.F46 v1tr.F46: (SEQ ID NO: 378) TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATAAATA

TABLE 15

<Composition of 10 mL Scale System>

-   -   Product obtained by the extension: 1 mL     -   MgCl₂: 2.5 mM     -   dNTPs: 0.25 mM     -   KCl: 50 mM     -   Tris-HCl (pH 9.0): 10 mM     -   Triton X-100: 10 μL     -   Primers: 250 nM (T7YGM.F46 v1tr.F46 and rCNSGGVSan13.R39)

Taq Polymerase

<PCR of 10 mL Scale System>

PCR (polymerase chain reaction) was performed, as shown in Table 16, by keeping at 95° C. for 40 seconds, keeping at 65° C. for 40 seconds, and then keeping at 72° C. for 40 seconds. Keeping at 95° C. for 40 seconds, keeping at 65° C. for 40 seconds and then keeping at 72° C. for 40 seconds were performed with 4 cycles.

It is to be noted that the following are the respective sequences of the primers T7YGM.F46 v1tr.F46 and rCNSGGVSan13.R39.

T7YGM.F46 v1tr.F46: (SEQ ID NO: 378) TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATAAATA rCNSGGVSan13.R39: (SEQ ID NO: 379) TTTCCGCCCCCCGTCCTAGCTTACCCCACCACTGTTACA

TABLE 16

As shown in FIG. 5, it was confirmed that a desired product was obtained by PCR.

(Purification of mRNA Library)

Preparation of an mRNA library was successfully achieved by performing in vitro transcription (4 mL scale) of the resulting PCR product with T7 RNA polymerase and purifying the products by electrophoresis under conditions of 12% denaturing-PAGE, 230V, and 70 minutes. As shown in FIG. 6 showing the gel of electrophoresis, it has been confirmed that a desired product was obtained, separated from a byproduct. Translation of the mRNA library thus obtained in a FIT system and contact with a prenylation enzyme as described above make it possible to introduce a prenyl group into the peptide of the peptide library obtained from the mRNAs.

Example 18: Modification Reaction of Artificial Cyclic Peptide with KgpF in Solid Phase

To prove that prenylating modification could be performed not only in a liquid phase but also in a solid phase, it was studied whether or not an artificial cyclic peptide could be modified with a KgpF enzyme immobilized onto the surface of magnetic beads via a tag.

(Immobilization of KgpF onto Magnetic Beads)

Dynabeads His-Tag Isolation and Pulldown (Invitrogen) was brought into contact with KgpF having a His-tag added N terminal and the KgpF was immobilized onto the magnetic beads. As shown in FIG. 7, with an increase in the amount of KgpF brought into contact with the beads, an immobilized amount of KgpF to the beads increased. When it exceeded a predetermined amount, immobilization reached saturation.

(Modification of Artificial Cyclic Peptide with KgpF Immobilized onto Magnetic Beads)

The artificial cyclic peptides shown in Table were synthesized under (Conditions 25) and were brought into contact with KgpF in solid phase under (Conditions 26). In addition, the same peptides were reacted in liquid phase under conditions shown in (Conditions 13) and (Conditions 14) except that the prenylation reaction time was changed to 14 hours.

(Conditions 25)

-   -   FIT system: v8s/v11.1     -   19aa-Met: 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   Reaction: 37° C., 60 min.     -   Scale: 2.5 μL scale

After the above reaction was performed, the reaction product was incubated at 25° C. for 12 minutes.

Then, a cyclization reaction was performed under the following conditions.

-   -   Translated mixture amount: 2.5 μL (mixture obtained by the above         reaction)     -   EDTA: 18 mM     -   Reaction: 42° C., 60 min.     -   Scale: 2.75 μL scale

(Conditions 26)

-   -   Translated mixture amount: 2.75 μL (mixture obtained under         (Conditions 25))     -   KOH: 6.0 mM     -   Mg(OAc)₂: 9.0 mM     -   Tris-HCl (pH 8.3) 15 mM     -   KgpF immobilized onto magnetic beads: 72 μM     -   DMAPP: 2 mM     -   Reaction: 4° C., 14 hours, rotation mixing

The solution obtained by the reaction was analyzed by MALDI-TOF-MS. As shown in the table, although the KgpF concentration is lower in solid phase, the modification efficiency is 100%, similarly to that in liquid phase (no peak of start material was found). This has revealed that modification can be achieved in solid phase similarly to that in liquid phase.

Shown in Table 17 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.

TABLE 17 The number of prenyl groups added SEQ Sequence Initiator template KgpF 0 1 2 3 ID Clone-V3 ClAc-(D)Tyr VWNVKIKPKTTKC Liquid 160 μM 1 5 N/A N/A 304 phase Clone-V3 ClAc-(D)Tyr VWNVKIKPKTTKC Solid  72 μM 1 5 N/A N/A 304 phase Clone-T2 ClAc-(D)Tyr LVRERENTWKVTC Liquid 160 μM 1 4 N/A N/A 308 phase Clone-T2 ClAc-(D)Tyr LVRERENTWKVTC Solid  72 μM 1 5 N/A N/A 308 phase

Example 19: Preparation of Compound Library and Screening

A library was prepared based on the design of the mRNA library shown in Example 17 in a manner similar to that of Example 17 except that the XXX portion was replaced with RYU.

A peptide-mRNA complex library was constructed from the above mRNA library according to the scheme shown in FIG. 8 and then, treated with a KgpF enzyme immobilized onto magnetic beads to construct a compound library including prenylated peptides.

Then, the compound library was provided for a step of screening cyclic peptides having interaction with an enzyme MetAP1 (Human) as in the scheme shown in FIG. 8. As a result, a library in which cyclic peptides binding to the MetAP1 had been enriched was obtained as shown in FIG. 9. Peptide sequences included in the resulting enriched library are shown in FIG. 10. It has been confirmed that with regards to some of the peptides shown in FIG. 10, peptides having these sequences are modified by prenylation as shown in Table 18 by bringing them into contact with KgpF under conditions shown in (Conditions 13) and (Conditions 14).

The above results have revealed that a compound library can be provided by the production method of the present invention using a prenylation enzyme.

Shown in Table 18 and Table 19 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.

TABLE 18 Sequence The number of prenyl added to groups added SEQ Sequence Initiator template C terminal 0 1 2 3 NO Met3 ClAc-(D)Tyr KRKAWICQTAVYC NSGGVS 2 5 N/A N/A 354 Met9 ClAc-(D)Tyr KRKNCLVHVWHQC NSGGVS 1 5 N/A N/A 355 Met11 ClAc-(D)Tyr HTDPFKAWHRRSC NSGGVS 1 5 N/A N/A 356 Met12 ClAc-(D)Tyr YIIWHRHRIMTVC NSGGVS 3 4 N/A N/A 357 Met13 ClAc-(D)Tyr VTWKGPYRWRVC NSGGVS 1 5 1 N/A 358

Example 19-1: Assessment of Peptides Obtained as a Result of Screening Against MetAP1 (Pulldown Assay on Binding Ability to MetAP1)

With regards to the prenylated cyclic peptides identified as a result of screening with MetAP1 as a target, their binding ability to the target was assessed by qualitative pulldown assay. After synthesis of Met3, Met9, Met11, Met12, Met13 and Met14 while displaying each of the sequences on mRNA, the Trp residue in the sequence was prenylated. The prenylated peptide-mRNA complex thus obtained was pulled down with magnetic beads and then, a recovered mRNA amount was determined by RT-qPCR. The results are shown in FIG. 11.

In the bar graph of FIG. 11, Negative bar indicates a pulldown amount when MetAP1-non-immobilized beads are used and Positive bar indicates a pulldown amount when MetAP1—immobilized beads are used. It has been confirmed that the pulldown amount is significantly higher in Positive than in Negative and the prenylated peptide sequence binds to MetAP1.

(Synthesis of Prenylated Cyclic Peptide by Using Fmoc Solid-Phase Synthesis and Prenylation with KgpF)

After chemical synthesis of respective cyclic peptides having sequences Met2 (SEQ ID NO: 361), Met3-1 (SEQ ID NO: 360), Met3a-L (SEQ ID NO: 362), Met11 (SEQ ID NO: 363) and Met13 (SEQ ID NO: 364) by Fmoc solid-phase synthesis, they were treated with KgpF in a test tube to prepare prenylated cyclic peptides. The prenylation was performed under the following Conditions 27.

(Conditions 27)

-   -   Scale: 1 mL     -   Cyclic peptide: 300 μM (in Met3-1, Met11, and Met13) or 1500 μM         (in Met2 and Met3a-L)     -   MgCl₂: 5.0 mM     -   KgpF: 16 μM (in Met11 and Met13)         -   or 75 μM (in Met2, Met3-1, and Met3a-L)     -   DMAPP: 1 mM     -   HEPES-KOH (pH 7.5) 50 mM     -   Reaction: 37° C., 20 hours

As shown in FIG. 12, it has been confirmed from MALDI-TOF-MS spectrum performed before and after the treatment with KgpF that intended prenylated cyclic peptides are obtained.

(MetAP1 Inhibition Test of Prenylated Cyclic Peptide)

A MetAP1 inhibition test was performed in vitro using the prenylated Met2, Met3a-L, and Met3-1 at concentrations of 1 μM, 5 μM or 10 μM in the presence of a MetAP1 cofactor Mn²⁺ or Co²⁺. In the test, enzymatic activity was measured by detecting probe fluorescence, that is, fluorescence emitted depending on the enzymatic activity of MetAP1. The measurement results of fluorescence intensity are shown in FIG. 13. As shown in FIG. 13, it has been confirmed that the prenylated Met2, Met3, and Met3a-L peptides could each inhibit the enzymatic activity of MetAP1 which was a target.

(Conditions)

-   -   Scale: 20 μL     -   Cyclic peptide: 1, 5, 10 μM     -   MnCl₂ or CoCl₂: 100 μM     -   MetAP1 enzymatic activity probe: 1 μM     -   MetAP1: 1 μM     -   Tris (pH7.6): 25 mM     -   NaCl: 75 mM     -   Reaction: 37° C., 120 min.

After the reaction, the fluorescence intensity of the probe was measured using a plate reader.

Example 20: Characteristics of Enzymatic Reaction of Prenylation Enzyme

Prenylation reaction was performed using the cyclic peptide shown below under conditions shown in (Conditions 27) in the presence or absence of Mg2+ at a concentration of EDTA adjusted to 0 mM, 1 mM or 10 mM. The cyclic peptides shown below were obtained by the synthesis using solid-phase synthesis.

(Conditions 27)

-   -   Scale: 5.7 μL     -   Cyclic peptide: 120 μL     -   MgCl₂: 5.0 mM (Mg(+); when used)     -   KgpF or OltF: 16 μM (KgpF) or 8.5 μM (OltF)     -   DMAPP: 2 mM     -   HEPES-KOH (pH7.5): 50 mM     -   Reaction: 37° C., 16 hours

The reaction products were analyzed by MALDI-TOF-MS. As a result, it has been found that the reaction proceeded dependently on Mg²⁺ when KgpF was used as a prenylation enzyme (refer to FIG. 14 and FIG. 15) and the reaction proceeded independently on Mg²⁺ when OltF was used (refer to FIG. 16).

Example 21: Prenylation of Cyclic Peptide Containing Non-Proteinogenic Amino Acid

A prenylation test with KgpF was performed using a cyclic peptide containing an amino acid in D-form as a non-proteinogenic amino acid. The cyclic peptide containing an amino acid in D-form (X in the following formula) is represented by the following formula.

The above artificial cyclic peptides containing an amino acid in D-form were obtained by translation and synthesis under the following (Conditions 28) in the artificial translation system developed by the present inventors. The translated mixtures thus obtained were then reacted (prenylated) under the following (Conditions 29).

(Conditions 28)

-   -   5aa-mix (K,R,A,W,C): 0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   D-aa-tRNA^(Pro1E2) _(CGG): 90 μM     -   Reaction: 37° C. and 60 minutes in artificial translation-system         solution     -   Scale: 2.5 μL scale

(Conditions 29)

-   -   Translated mixture amount: 2.5 pL (mixture obtained under         (Conditions 28))     -   KgpF: 16 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 20 hours

The reaction products were analyzed by MALDI-TOF-MS. As a result, it has been confirmed that the respective artificial cyclic peptides containing, as X, Ile in D-form, Phe in D-form, Trp in D-form and Tyr in D-form were modified by prenylation (refer to FIG. 17).

Example 22: Prenylation of Non-Proteinogenic Trp in Cyclic Peptide Containing the Trp

By using a cyclic peptide containing a non-proteinogenic amino acid Trp, a prenylation test of the Trp with OltF or KgpF was performed. The cyclic peptide used is as follows. In the formula, X is Trp in D-form.

The above artificial cyclic peptide containing Trp in D-form was obtained by translation and synthesis under the following (Conditions 30) in the artificial translation system developed by the present inventors. The translated mixture thus obtained was then reacted (prenylated) under the following (Conditions 31) at 37° C. for 20 hours.

(Conditions 30)

-   -   19aa-Mix (19 kinds of proteinogenic amino acids other than M)]:         0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 90 μM     -   D-Trp-tRNA^(Pro1E2) _(CGG): 90 μM     -   Reaction: 37° C. and 60 minutes in an artificial         translation-system solution.     -   Scale: 2.5 μL scale

(Conditions 31)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 30))     -   KgpF or OltF: 170 μM     -   DMAPP: 2 mM     -   Reaction: 37° C., 20 hours

The reaction product was analyzed by MALDI-TOF-MS. As a result, it has been confirmed that the artificial cyclic peptide containing, as X, Trp in D-form was modified by prenylation (refer to FIG. 18).

Example 23: Prenylation of Trp Present at N Terminal

The cyclic peptide shown below was obtained by translation and synthesis under the following (Conditions 32) in the artificial translation system developed by the present inventors. The translated mixture thus obtained was then reacted (prenylated) under the following (Conditions 33).

(Conditions 32)

-   -   19aa-mix (19 kinds of proteinogenic amino acids other than M)]:         0.5 mM     -   ClAc-D-Tyr-tRNA^(fMet) _(CUA): 50 μM     -   Reaction: 37° C. and 30 minutes in an artificial         translation-system solution.     -   Scale: 2.5 μL scale

(Conditions 33)

-   -   Translated mixture amount: 2.5 μL (mixture obtained under         (Conditions 32))     -   OltF: 100 μM     -   DMAPP: 2.1 mM     -   Reaction: 37° C., 18 hours     -   Scale: 5.7 μL scale

The reaction product was analyzed by MALDI-TOF-MS. As a result, it has been found that a Trp at two positions, that is, at the N-terminal position and in the sequence could be prenylated by using OltF as a prenylation enzyme (refer to FIG. 19).

In a manner similar to that described above, prenylation of AY015 (SEQ ID NO: 372), AY016 (SEQ ID NO: 373), AY017 (SEQ ID NO: 374), AY018 (SEQ ID NO: 375), AY019 (SEQ ID NO: 376) and AY020 (SEQ ID NO: 377) (refer to FIG. 20) was performed with OltF. It has been confirmed that a Trp at the N terminal position was prenylated. 

1. A method of producing a peptide library comprising a prenylated peptide, comprising a step of bringing a peptide library comprising a peptide having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.
 2. The method of producing a peptide library according to claim 1, wherein the prenylation enzyme is at least one or more selected from the group consisting of Enzyme 1 to Enzyme 21 and homologs thereof.
 3. The method of producing a peptide library according to claim 1, wherein the prenylation enzyme consists of any amino acid sequence of the following (1) to (3): (1) an amino acid sequence represented by any of SEQ ID NOS: 1 to 56, (2) an amino acid sequence with one or more amino acid deletions, substitutions or additions in an amino acid sequence represented by any of SEQ ID NOS: 1 to 56, and (3) an amino acid sequence having 80% or more homology with an amino acid sequence represented by any of SEQ ID NOS: 1 to
 56. 4. The method of producing a peptide library according to claim 1, wherein the peptide library comprises a cyclic peptide.
 5. The method of producing a peptide library according to claim 4, wherein the cyclic peptide has a cyclic structure formed by bonding of two amino acid residues through a disulfide bond, peptide bond, alkyl bond, alkenyl bond, ester bond, thioester bond, ether bond, thioether bond, phosphonate ether bond, azo bond, C—S—C bond, C—N—C bond, C═N—C bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, or triazole bond.
 6. The method of producing a peptide library according to claim 1, comprising, prior to the prenylation step, a step of translating an mRNA library in a cell-free translation system and preparing a peptide library comprising a peptide having an amino acid sequence containing at least one Trp or derivative thereof.
 7. A method of producing a peptide-genotype complex library, comprising: a step of preparing a peptide-genotype library comprising a complex between a genotype and a peptide, and a step of bringing the peptide-genotype library into contact with a prenylation enzyme and prenylating at least one Trp or derivative thereof.
 8. The method of producing a peptide-genotype complex library according to claim 7, wherein: the step of preparing the peptide-genotype library comprises a step of preparing a peptide-mRNA library by mRNA display method; and the step of preparing the peptide-mRNA library comprises: a step of binding a puromycin to a 3′-end of each mRNA of an mRNA library to produce a puromycin-bound mRNA library, and a step of translating the puromycin-bound mRNA library in a cell-free translation system and preparing a peptide-mRNA library comprising a peptide having an amino acid sequence containing at least one Trp or derivative thereof.
 9. A screening method for identifying a peptide binding to a target material, comprising: a step of bringing the peptide library prepared by the production method as claimed in claim 1 into a target material, and a step of selecting a peptide binding to the target material.
 10. A method of producing a prenylated peptide, comprising a step of bringing a peptide having an amino acid sequence containing at least one Trp or derivative thereof into a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.
 11. A method of producing a prenylated peptide according to claim 10, comprising a step of, prior to the prenylation step, synthesizing a peptide having an amino acid sequence containing at least one Trp or derivative thereof by chemical synthesis.
 12. The method of producing a peptide library according to claim 1, wherein: the peptide is a peptide linked to genotype, and the method further comprises a step of preparing a peptide library comprising a peptide having an amino acid sequence containing at least one Trp or derivative thereof and linked to genotype.
 13. The method of producing a peptide library according to claim 12, wherein: the step of preparing the peptide library comprises a step of preparing a peptide-mRNA library by mRNA display method; and the step of preparing the peptide-mRNA library comprises: a step of binding a puromycin to a 3′-end of each mRNA of an mRNA library to produce a puromycin-bound mRNA library, and a step of translating the puromycin-bound mRNA library in a cell-free translation system and preparing a peptide-mRNA library comprising a peptide having an amino acid sequence containing at least one Trp or derivative thereof.
 14. A screening method for identifying a peptide binding to a target material, comprising: a step of bringing the peptide-genotype complex library prepared by the production method as claimed in claim 7 into a target material, and a step of selecting a peptide binding to the target material.
 15. A screening method for identifying a peptide binding to a target material, comprising: a step of bringing the peptide-genotype complex library prepared by the production method as claimed in claim 8 into a target material, and a step of selecting a peptide binding to the target material.
 16. A screening method for identifying a peptide binding to a target material, comprising: a step of bringing the peptide library prepared by the production method as claimed in claim 12 into a target material, and a step of selecting a peptide binding to the target material.
 17. A screening method for identifying a peptide binding to a target material, comprising: a step of bringing the peptide library prepared by the production method as claimed in claim 13 into a target material, and a step of selecting a peptide binding to the target material.
 18. The method of producing a peptide library according to claim 2, wherein the peptide library comprises a cyclic peptide.
 19. The method of producing a peptide library according to claim 3, wherein the peptide library comprises a cyclic peptide.
 20. The method of producing a peptide library according to claim 2, comprising, prior to the prenylation step, a step of translating an mRNA library in a cell-free translation system and preparing a peptide library comprising a peptide having an amino acid sequence containing at least one Trp or derivative thereof. 